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A Guide to Polyacrylamide and Detection

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Protein )

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Fluorescent Gel Stain Coomassie Stain Coomassie ™ ™ Fluorescent Gel Stain ™ Pour the Stacking Gel

General Tips for Sample PreparationSample for General Tips Standard Assay Protocol (5 ml) Microcentrifuge Assay Tube Protocol ml) (1.5 General Protocols: SDS-PAGE Silver (Bio-Rad Silver Stain) Fractions from from Fractions Protein Sample Buffers Running Buffers Buffer Components Total Protein Staining Protein Total Sample QuantitationSample ( Performing Electrophoresis Sample Preparation BuffersSample Reagents Casting Gel Sample PreparationSample Molecular Weight Estimation Molecular Weight Handcasting Polyacrylamide Gels

Glossary References and Related Reading Part Methods II: Protocols Gels Single-Percentage Pour the Resolving Gel Gels Gradient Bio-Safe Oriole Flamingo Part III: Troubleshooting Preparation Sample Gel Casting and Sample Loading Electrophoresis Staining Protein Total Separation of Evaluation Part IV: Appendices Ordering Information (Cell Disruption) (Cell Lysis Solubilization Protein Cells Preparation PAGE for Human Cells Cultured Suspension Tissue CulturedCells Monolayer Leaves Mammalian Cultures Plant Microbial Buffer Formulations

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High-Throughput Stainers High-Throughput

Stands

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Separations Under Constant Voltage Constant Under Separations Constant CurrentSeparations Under Power Constant Under Separations

Specific Protein Stains Protein Specific Gradient Formers Selecting Power Supply Settings Total Protein Stains Protein Total Multi-Casting Chambers Useful Equations Useful Joule Heating AffectingOther Factors Electrophoresis Laemmli (Tris-HCl) Tris-Acetate Tris-Tricine Zymogram Estimation (Size) Molecular Weight Quantitation Percentage Handcast vs. Precast Format (Size and Comb Type) Imaging Systems Imaging Imaging Software Premade Buffers and Reagents Buffers and Premade Dodeca

Chapter 6 and Analysis Stains Protein Imaging Analysis Chapter 7 Downstream Applications Blotting (Immunoblotting)Western Drying Gel Electroelution Spot Excision (Cutting) Polyacrylamide Gels Polyacrylamide Buffer Systems and Gel Chemistries Handcasting Gels for Products Electrophoresis Performing 5 Chapter System Setup Conditions Running Conditions Running for Guidelines General Gel Disassembly and Storage Bis-Tris IEF Polymerization

AnyGel

6 6 5 9 5 11 12 12 12 15 13 13 13 13 19 19 10 10 10 27 21 21 21 21 21 23 22 22 26 26 26 20 20 20 25

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Discontinuous Native PAGE Native Discontinuous Other Types of PAGE Natural Standards Standards Natural Recombinant Standards Recombinant Protein Assays 2-D Electrophoresis2-D Isoelectric (IEF) Focusing Buffers and Salts Solubilization Protein for Solutions Common Polyacrylamide Gel Electrophoresis (PAGE) Electrophoresis Cells Applications PAGE for Supplies Power Reducing AgentsReducing AgentsChaotropic

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Protein Standards Protein Chapter 3 Sample Preparation for for Preparation Sample 3 Chapter Electrophoresis General Considerations Cell Disruption Solubilization Protein Removal of Interfering Substances Products for Sample Preparation SampleQuantitation (Protein Assays) Chapter 4 Reagent Selection and General Considerations Part Theory I: Selection Product and Chapter 1 Overview Electrophoresis Works Protein How Workflow General Considerations and Electrophoresis Methods Protein 2 Chapter and Instrumentation Electrophoresis Methods Protein SDS-PAGE Blue Native (BN-PAGE) PAGE Zymogram PAGE Electrophoresis Cells and Power Supplies Preparation

2 Electrophoresis Guide

TABLE OF CONTENTS 5 Theory and Product Selection Theory Product and Chapter 1: OverviewChapter 1:

Overview is the electrophoresis Protein an within of movement widely and Popular field. electric commonly most is it research, in used the for proteins separate to used purposes analysis of and purification. overview a brief provides chapter This behind theory workflow and the of electrophoresis. protein PART I Theory and Product Selection CHAPTER 1

4 Electrophoresis Guide

TABLE OF CONTENTS 7 Theory and Product Selection Theory Product and Chapter 1: OverviewChapter 1:

during separation. during Method SelectionMethod for electrophoresis. for imaging equipment. imaging Sample Preparation resolution and throughput. and resolution Gel and Buffer Preparation electrophoresis performed. electrophoresis Performing Electrophoresis Protein Detection and Analysis Select a visualization technique that that technique visualization Select a depends on the gel type and type of Whether handcast or precast, the gel the appropriate electrophoresis method. method. electrophoresis appropriate the The protein sample may be prepared from Gels are placed in the electrophoresis cell, Consider the experimental goals in selecting matches sensitivity requirements and available available and requirements sensitivity matches while maintaining the temperature the of system buffer is added, and samples are loaded. Select protein at concentration a and in a buffer suitable Instrumentation selection depends on the desired desired the on depends selection Instrumentation a biological sample, or it may come from a step in type used should suit the properties the of purification workflow. In either case, prepare the running conditions that provide optimum resolution under investigation, the desired analysis technique, and overall goals the of experiment. Buffer selection Fig. 1.2. Protein electrophoresis workflow. electrophoresis workflow. Protein 1.2. Fig. Protein Electrophoresis Workflow Electrophoresis Protein – Power supply + Cathode – – + Electrodes + + – Anode The electrophoresis workflow involves1.2) the (Figure instrumentation, method, appropriate the of selection and reagents for the intended experimental goal. Once proteins are separated, they are available for a number downstream of applications, including enzymatic assays, further purification, transfer detection immunological for membrane a to (immunoblotting or western blotting), and elution and digestion for mass spectrometric analysis. Fig. 1.1. Movement of proteins during electrophoresis. during proteins of Movement 1.1. Fig. Workflow General Considerations and

/Vsec) is 2

(Figure 1.1). Proteins come in a wide range sizes of (Figure 1.1). and shapes and have charges imparted them to the by dissociation constants their of constituent amino acids. As a result, proteins have characteristic purpose the for exploited be can that rates migration separation.of Protein electrophoresis can be performed in either liquid or gel-based media and can also be used move proteins to from one medium anotherto (for example, in blotting applications). Over the last 50 years, electrophoresis techniques have evolved as refinements have been madeto the buffer systems, instrumentation, and visualization used be can electrophoresis Protein used. techniques for a variety applications of such as purifying proteins, assessing protein purity (for example, at various stages data gathering separation), chromatographic a during determining or expression, protein of regulation the on protein size, isoelectric point and (pI), enzymatic activity.In fact, a significant numbertechniquesof (IEF), isoelectric focusing gel electrophoresis, including two-dimensional and (blotting), transfer electrophoretic electrophoresis(2-D) can be grouped under the term Though 2010). (Rabilloud electrophoresis” “protein some information is provided about these methods in the following chapters, this guide focuses on the one- polyacrylamide in proteins of separation dimensional (PAGE). polyacrylamide or electrophoresis gel gels, governed a complex by relationship between the electrophoresis the both of characteristics physical system and the proteins. Factors affecting protein electrophoresis include the strength the of electric field,temperature the of the system, the pH, type, and concentration the of buffer as well as the size, shape, and charge the of proteins (Garfin1990) How Protein Electrophoresis Works Protein How The term electrophoresis refers the movement to of chargedof molecules in response an electric to field, resulting in their separation. In an electric field, towardproteins the move electrode opposite of charge. The at which rate they move (migration in units rate, cm of

6 bulletin 2651 bulletin and ProductManual, : A Methods Methods A Proteomics: and Detection, and for Electrophoresis 2-D A Guide Transfer to 2895 bulletin Protein Blotting Guide, Related Literature Related Electrophoresis Guide

TABLE OF CONTENTS 9 Theory and Product Selection Theory Product and Chapter 2: Protein Electrophoresis Protein 2: andChapter Methods Instrumentation

Consider the experimental goals in in goals experimental the Consider electrophoresis appropriate selecting the instrumentation of selection method; volume and number the on depends and resolution, desired samples, of the describes chapter This throughput. systems and techniques common most in use today. CHAPTER 2 Protein Electrophoresis Methods and Instrumentation

8 Electrophoresis Guide

TABLE OF CONTENTS Electrophoresis Guide Chapter 2: Protein Electrophoresis Methods and Instrumentation Theory and Product Selection

Protein Electrophoresis Methods Two types of buffer systems can be used: Related Literature By choosing suitable separation matrices and ■■ Continuous buffer systems use the same buffer corresponding buffer systems, a range of experimental (at constant pH) in the gel, sample, and electrode Stacking gel Gel Electrophoresis: objectives can be met using protein electrophoresis reservoirs (McLellan 1982). Continuous systems are 4%T*, pH 6.8 Separation of Native Basic (Zewart and Harrington 1993). not common in protein separations; they are used Proteins by Cathodic, Polyacrylamide Gel Electrophoresis (PAGE) mostly for nucleic acid analysis Discontinuous Polyacrylamide Gel Electrophoresis, When electrophoresis is performed in acrylamide or ■■ Discontinuous buffer systems use a gel separated bulletin 2376 gels, the gel serves as a size-selective sieve into two sections (a large-pore stacking gel on top of during separation. As proteins move through a gel in a small-pore resolving gel, Figure 2.2) and different

response to an electric field, the gel’s pore structure buffers in the gels and electrode solutions (Wheeler Resolving gel allows smaller proteins to travel more rapidly than larger et al. 2004) 7.5%T to 15%T, pH 8.8 proteins (Figure 2.1). For protein separation, virtually In gel electrophoresis, proteins do not all enter the all methods use polyacrylamide as an anticonvective, gel matrix at the same time. Samples are loaded sieving matrix covering a protein size range of into wells, and the proteins that are closer to the gel 5–250 kD. Some less common applications such as enter the gel first. In continuous systems, the uniform immunoelectrophoresis and the separation of large separation matrix yields protein bands that are diffuse proteins or protein complexes >300 kD rely on the larger and poorly resolved. In discontinuous systems, on the pore sizes of agarose gels. other hand, proteins first migrate quickly through the In most PAGE applications, the gel is mounted between large-pore stacking gel and then are slowed as they two buffer chambers, and the only electrical path enter the small-pore resolving gel. As they slow down, Fig. 2.2. Migration of proteins and buffer in a denaturing discontinuous PAGE system. A, Denatured sample proteins are loaded between the two buffers is through the gel. Usually, the they stack on top of one another to form a tight band, into the wells; B, Voltage is applied and the samples move into the gel. The chloride ions already present in the gel (leading ions) run faster

TABLE CONTENTS OF gel has a vertical orientation, and the gel is cast with which improves resolution. Discontinuous systems also than the SDS-bound proteins and form an ion front. The glycinate ions (trailing ions) flow in from the running buffer and form a front behind a comb that generates wells in which the samples are use ions in the electrophoresis buffer that sandwich the proteins; C, A voltage gradient is created between the chloride and glycinate ions, which sandwich the proteins in between them; D, The proteins are stacked between the chloride and glycinate ion fronts. At the interface between the stacking and resolving gels, the percentage applied (Figure 2.1). Applying an electrical field across the proteins as they migrate through the gel, and this of acrylamide increases and the pore size decreases. Movement of the proteins into the resolving gel is met with increased resistance; E, The the buffer chambers forces the migration of protein into tightens the protein bands even more (Figure 2.2). smaller pore size resolving gel begins to separate the proteins based on molecular weight only, since the charge-to-mass ratio is equal in all and through the gel (Hames 1998). Discontinuous buffer systems provide higher resolution the proteins of the sample; F, The individual proteins are separated into band patterns ordered according to their molecular weights. than continuous systems, and varying the buffers used * %T refers to the total monomer concentration of the gel (see Chapter 4 for more information). Cathode in the sample, gel, and electrode chambers creates a variety of discontinuous buffer systems that can be Nevertheless, native PAGE does allow separation of As a result, the rate at which SDS-bound protein used for a variety of applications. proteins in their active state and can resolve proteins of migrates in a gel depends primarily on its size, enabling Discontinuous Native PAGE the same molecular weight. molecular weight estimation. Well Buffer The original discontinuous gel system was developed SDS-PAGE Larger by Ornstein and Davis (Ornstein 1964, Davis 1964) The original Laemmli system incorporated SDS in (high MW) To overcome the limitations of native PAGE systems, protein for the separation of serum proteins in a manner the gels and buffers, but SDS is not required in the Laemmli (1970) incorporated the sodium that preserved native protein conformation, subunit gel. SDS in the sample buffer is sufficient to saturate dodecyl sulfate (SDS) into a discontinuous denaturing Protein band interactions, and biological activity (Vavricka 2009). proteins, and the SDS in the cathode buffer maintains buffer system, creating what has become the most Smaller Anode In such systems, proteins are prepared in nonreducing, the SDS saturation during electrophoresis. Precast gels Direction of protein migration protein of Direction (low MW) popular form of protein electrophoresis, SDS-PAGE. (manufactured gels such as Bio-Rad’s Mini-PROTEAN® protein nondenaturing sample buffer, and electrophoresis ™ is also performed in the absence of denaturing and When proteins are separated in the presence of SDS and Criterion gels) do not include SDS and so can reducing agents. and denaturing agents, they become fully denatured be used for either native or SDS-PAGE applications. and dissociate from each other. In addition, SDS binds A range of gel and buffer combinations can be used for Data from native PAGE are difficult to interpret. Since Gel noncovalently to proteins in a manner that imparts: native and SDS-PAGE, each with its own advantages the native charge-to-mass ratio of proteins is preserved, (see Chapter 4 for more details). ■■ An overall negative charge on the proteins. Fig. 2.1. Schematic of electrophoretic protein separation in a protein mobility is determined by a complex combination Since SDS is negatively charged, it masks the polyacrylamide gel. MW, molecular weight. of factors. Since protein-protein interactions are retained O – S O O intrinsic charge of the protein it binds O O– during separation, some proteins may also separate O S O O as multisubunit complexes and move in unpredictable ■■ A similar charge-to-mass ratio for all proteins in a SDS mixture, since SDS binds at a consistent rate of O–Na+ ways. Moreover, because native charge is preserved, O S O O proteins can migrate towards either electrode, 1.4 g of SDS per 1 g protein (a stoichiometry of O –

S O O– O O O depending on their charge. The result is that native about one SDS molecule per two amino acids) S O O PAGE yields unpredictable separation patterns that ■■ A long, rod-like shape on the proteins instead of a are not suitable for molecular weight determination. complex tertiary conformation (Figure 2.3)

Fig. 2.3. Effect of SDS on the conformation and charge of a protein.

10 11 Electrophoresis Guide Chapter 2: Protein Electrophoresis Methods and Instrumentation Theory and Product Selection

Isoelectric Focusing (IEF) Bio-Rad’s PROTEAN® i12™ IEF system provides Automated Electrophoresis IEF combines the use of an electric field with a pH individual lane control for up to 12 IPG strips, making gradient to separate proteins according to their pI. it possible to run different sample types, different pH Automated electrophoresis systems use microfluidic The types of data available include the following: It offers the highest resolution of all electrophoresis gradients, and multiple protocols at the same time. IEF technology to integrate electrophoretic separation, n Protein size (or mass, in kD) techniques (Westermeier 2004). can be run under either native or denaturing conditions. fluorescence detection, and data analysis within a ■■■Protein ■ concentration (in ng/μl): relative and Native IEF retains protein structure and enzymatic single platform. Using much smaller sample and When a protein moves through a pH gradient, its net absolute concentration activity. However, denaturing IEF is performed in reagent quantities and far less time than standard charge changes in response to the pH it encounters. ■■■Consider ■ protein purity (% of the total sample) as the presence of high concentrations of urea, which analysis methods, automated electrophoresis Under the influence of an electric field, a protein in a an alternative dissociates proteins into individual subunits and systems like Bio-Rad’s Experion™ system can pH gradient migrates to a pH where its net charge is abolishes secondary and tertiary structures. Whereas be used in applications such as purity and yield The Experion system provides single-step zero (the protein’s pI). If the protein moves out of that native IEF may be a more convenient option because assessment in and protocol quantitation and size determination (sizing) of protein position, it acquires a charge and is forced back to it can be performed with a variety of precast gels, optimization workflows. However, samples cannot samples useful for quality control, protein purity the zero-charge position (Figure 2.4). This focusing is denaturing IEF often offers higher resolution and is more be recovered from automated separations, and and stability analysis, protocol optimization, and responsible for the high resolution of IEF. pI values of suitable for the analysis of complex protein mixtures. downstream applications are thus not possible. evaluation of recombinant protein expression. proteins usually fall in the range of pH 3–11. Related Literature 2-D Electrophoresis The Experion system employs LabChip microfluidic pH 3 4 5 6 7 8 9 10 The sequential application of different electrophoresis + technology to automate complete protein and 4 + NH3 NH NH2 NH2 NH3 2 NH2 techniques produces a multi-dimensional separation. 2-D Electrophoresis for nucleic acid electrophoresis and analyze up to Proteomics: A Methods and The most common 2-D technique (O’Farrell 1975) Product Manual, 12 samples in a single 30–40 min automated step COOH COOH COOH COOH COOH COO- subjects protein samples first to denaturing IEF on - bulletin 2651 (analysis of a single sample is accomplished in as COOH COOH COO a tube gel or IPG gel strip (for separation by pI), then little as 90 sec). Net Charge 1 to SDS-PAGE for further separation by molecular Fig. 2.4. Isoelectric focusing. A protein is depicted in a pH Protein analysis with the Experion system is weight. High-resolution 2-D methods enable separation

TABLE CONTENTS OF gradient in an electric field. A pH gradient formed by ampholyte achieved with the Experion Pro260 analysis kit, molecules under the influence of an electric field is indicated. of thousands of polypeptides in a single slab gel. which supplies the microfluidic chips, reagents, The gradient increases from acidic (pH 3) at the anode to basic The resulting spots can be visualized by gel staining, (pH 10) at the cathode. The hypothetical protein in the drawing bears or they can be transferred to a membrane support and instructions required to separate and analyze a net charge of +2, 0, or –2, at the three positions in the pH gradient 10–260 kD proteins under denaturing conditions. 2 3 shown. The electric field drives the protein toward the cathode when for total protein staining or analysis with specific The sensitivity of the Experion Pro260 analysis kit is it is positively charged and toward the anode when it is negatively detection. For more details, refer to 2-D charged, as shown by the arrows. At the pI, the net charge on the Electrophoresis for Proteomics (bulletin 2651). Links comparable to that of colloidal Coomassie (Brilliant) protein is zero, so it does not move in the field. The protein loses Blue staining of SDS-PAGE gels. protons as it moves toward the cathode and becomes progressively 5 less positively charged. Conversely, the protein gains protons as Electrophoresis Cells and Power Supplies Mini Format 1-D Experion software converts a fluorescent signal a it moves toward the anode and becomes less negatively charged. Electrophoresis Cells Electrophoresis Systems into an electropherogram (a plot of fluorescence When the protein becomes uncharged (pI), it ceases to move in the Vertical electrophoresis cells are plastic boxes with b field and becomes focused. vs. time) and a densitometric gel-like image, or anode and cathode buffer compartments that contain Mini-PROTEAN Precast Gels The Experion system. The system includes: 1, automated virtual gel. Each lane in the virtual gel corresponds electrodes (Figure 2.5). The electrodes (typically electrophoresis station; 2, priming station; 3, vortex station Two methods are used to generate a stable, continuous Mini-PROTEAN Tetra Cell to a different sample, and the peaks in the used only for nucleic acid analysis; 4, system operation and pH gradient between the anode and cathode: platinum wire) connect to a jack attached to a electropherogram correspond to bands in the virtual data analysis tools (software); and 5, analysis kits, which power supply. The gels are held vertically between Mini-PROTEAN 3 Dodeca Cell include the chips (a) and reagents (b) for protein (Pro260 kit), ■■ Carrier ampholytes — heterogeneous mixtures gel. The results of data analysis are also presented the electrode chambers during electrophoresis standard-sensitivity RNA (StdSens kit), high-sensitivity RNA of small (300–1,000 Da) conductive polyamino- Midi Format 1-D in a table at the end of analysis. (HighSens kit), and DNA (DNA 1K and 12K kits) analyses. (Andrews 1986). polycarboxylate compounds that carry multiple Electrophoresis Systems charges with closely spaced pI values. When voltage Electrodes Criterion Precast Gels Other Types of PAGE sizes; the protein complexes become focused at the is applied across an ampholyte-containing solution Links Blue Native PAGE (BN-PAGE) corresponding pore size limit (Nijtmans et al. 2002, or gel, the ampholytes align themselves according Criterion Cell BN-PAGE is used to separate and characterize large to their pIs and buffer the pH in their proximity, Reisinger and Eichacker 2008). Criterion Dodeca Cell Experion Automated protein complexes in their native and active forms. establishing a pH gradient. Ampholytes can be used Zymogram PAGE Electrophoresis System Originally described by Schägger and von Jagow in gels (for example, tube gels or vertical gels) or in Large-Format 1-D Zymogram PAGE is used to detect and characterize Lid (1987), this technique relies on the solubilization of solution (for example, liquid-phase IEF) Electrophoresis Systems Experion Protein collagenases and other within the gel. Analysis Kits protein complexes with mild, neutral detergents and ■■ Gels are cast with gelatin or casein, which acts as a Immobilized pH gradients (IPG) strips — formed PROTEAN II xi Cell the binding of negatively charged Coomassie (Brilliant) by covalently grafting buffering groups to a Coomassie Stains substrate for the that are separated in the Blue G-250 stain to their surfaces. This imparts a high PROTEAN II XL Cell gel under nonreducing conditions. The proteins are polyacrylamide gel backbone. A gradient of different Coomassie Brilliant charge-to-mass ratio that allows the protein complexes run with denaturing SDS in order to separate them buffering groups generates a stable pH gradient that PROTEAN II xi and XL Multi-Cells Blue G-250 Stain to migrate to the anode as they do in SDS-PAGE. by molecular weight. After renaturing the enzymes can be tailored for different pH ranges and gradients Coomassie Blue does not, however, denature and Running PROTEAN Plus Dodeca Cell Coomassie Brilliant and then allowing them to break down the substrate, (Bjellquist et al. 1982) Gel box dissociate protein complexes the way SDS does. High- module Blue R-250 Stain zymogram gels are stained with Coomassie (Brilliant) PROTEAN i12 IEF System resolution separation is achieved by electrophoresis Fig. 2.5. Components of a vertical electrophoresis cell. Blue R-250 stain, which stains the substrate while into an acrylamide gradient with decreasing pore leaving clear areas around active proteases.

12 13 15 Related Literature Related PowerPac Basic 300 V Power 2881 bulletin Flier, Supply High-Current HC PowerPac Power Supply Flier, 2882 bulletin PowerPac Universal Power Brochure, Supply 2885 bulletin PowerPac HV Power Supply Brochure, bulletin 3189 Links ElectrophoresisPreparative Power Supplies PowerPac Universal Power Supply High-Current HC PowerPac Power Supply PowerPac HV High-Voltage Power Supply PowerPac Basic Power Supply Model 491 Prep Cell and Mini Prep Cell Rotofor and Mini Rotofor Cells Cell MicroRotofor Theory and Product Selection Theory Product and

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PowerPac Basic Power Supply cell, PowerPac HV High-Voltage Power Supply Power High-Voltage HV PowerPac ™ PowerPac Universal Power Supply PowerPac HC High-Current Power Supply Use the PowerPac Basic or PowerPac HC high- vertical mini-format for supply power current applications PAGE Use the PowerPac HV high-voltage or PowerPac vertical large-format for supply power Universal applications PAGE Use the PowerPac HC power supply for applications that require high currents, with such the as PAGE Dodeca cells high-throughput fractionate proteins in free solution according to produces technique nondenaturing This pI. their fractions that can be collected, pooled, and even Fractionation further purification. for refractionated proteins particularly for is useful IEF liquid-phase by that do not separate well in gel-based IEF media. The pH gradient used for separation is generated ampholytes,by allowing a continuous and formed. be to gradient pH customizable Liquid-Phase IEF Rotofor the as such devices, IEF Liquid-phase Combination Approaches (2-D Separations) (2-D Approaches Combination Preparative can be IEF and combined PAGE (for separation on multiple dimensions) for even separation. greater cell, mini Rotofor cell, and MicroRotofor and cell, Rotofor mini cell, ■ ■ ■ Fig. 2.6. PowerPac power supplies. ■ ■ ■ of powerof supply applicationsfor PAGE usually depends on the size and number gels of being 2.2 run. Table supplies power PowerPac Bio-Rad the compares applications. vertical electrophoresis recommended for

cell

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HC HC HC HC HC HC HC HC Universal HC HC or Universal or HC Basic HC or Basic or HC Universal or HV Universal or HV Universal or HC Universal or HC PowerPac Power Supply Chapter 2: Protein Electrophoresis Protein 2: andChapter Methods Instrumentation Plus Dodeca ®

power supplies selection guide. selection supplies power

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Preparative electrophoresis techniques separate separate techniques electrophoresis Preparative large amounts protein of (nanogram gram to quantities) for the purposes purification of or complexity). sample reduce (to fractionation The same principles that are applied for analytical work can be applied for preparative work. PAGE canPreparative be accomplished PAGE using a standard slab gel or special instrumentation. With the slab gel, a single preparative or “prep” well is cast, which allows a large volume a single of sample to be applied within one well. With this approach, the separated protein is retained within the gel for further electroelution). by example, (for purification or analysis electrophoresis gel continuous-elution Alternatively, using the Model prep cell 491 or mini prep cell yields high-resolution separations and proteins in liquid fractions, ready for downstream use. Preparative ElectrophoresisPreparative allows maximum throughput with the capability to gels at a timerun12 up to and 20 x 20.5 cmand for the x 20.5 20 PROTEAN Plus System) and offer maximum resolution. The PROTEAN II system provides a choice glass of plates, spacer, and sandwich clamps cast to two gel lengths: or cm. The 20 PROTEAN16 Trans-Blot SD cell SD Trans-Blot Trans-Blot Plus cell Trans-Blot cell Trans-Blot Criterion blotter Criterion PROTEAN Plus Dodeca cell Blotting Western cell Trans-Blot Mini Criterion cell PROTEAN II xi cell PROTEAN II XL cell Electrophoresis High-Throughput Mini-PROTEAN 3 Dodeca cell Mini-PROTEAN Tetra cell Tetra Mini-PROTEAN cell Dodeca Criterion xi/XL multi-cell II PROTEAN O’Farrell Second Dimension (SDS) Dimension Second O’Farrell Apparatus (SDS), Laemmli Technique and Recommended and Technique Protein DNA/RNA Wire electrodes electrodes Plate transfer High-intensity Wire electrodes electrodes Plate

Power Supplies for PAGE Applications PAGE for Supplies Power Power supplies are available meet to the power requirements numerous of applications. The choice 2.2. PowerPac Table

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II system ® cell (for running ™ 18.5 x 20.5 cm 18.5 x 20.520.0 cm x 20.520.0 cm dimension of 2-D electrophoresis 2-D of dimension equipment casting Plus PROTEAN PROTEAN Plus Dodeca cell Plus Trans-Blot run gels) up 12 to second the for Specifically Offers maximum resolution in a range longest the and gel single of separation (with the ability to PROTEAN Plus System 3 Dodeca ® system includesthe Criterion cell

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cell (for 1–12 gels); both cells gels); are cell (for 1–12 ™

compatible with Criterion precast gels precast Criterion with compatible Dodeca up to 12 gels); both cells gels); are compatible 12 up to with gels precast Mini-PROTEAN capacity four up of to gels) and the high-throughput Mini-PROTEAN Midi-format cm systems — accommodate x 8.7 13.3 gels and offer rapid runs with more samples per gel and enhanced separation over mini-format gels. Criterion The Large-format systems — accommodate large gels cm for thePROTEAN x 18.3 20 to (up (for 1–2 gels) and the high-throughput Criterion high-throughput the and gels) 1–2 (for ■ ■ ■ ■

® PROTEAN II xi/XL PROTEAN II xi/XL multi-cells 20.0 x 18.3 cm x 18.3 20.0 Large-format gel system offers greater resolution over formats smaller Can accommodate up 4 gels to and is available in xi or XL formats for running a variety of gel sizes running for available is Multi-cell up 6 gels to PROTEAN II casting plates PROTEAN II System Trans-Blot cell Trans-Blot cell Plus Trans-Blot cell SD Trans-Blot

Criterion Criterion Dodeca Criterion gels: precast Criterion cm x 0.1 x 8.7 13.3 15.0 x 10.6 cm x 10.6 15.0 Fast setup with drop-in gel and cell design (precast or handcast) or (precast design cell Run precast 1–2 Criterion or cell Criterion the in gels handcast gelsand up 12 to in the Criterion cell Dodeca buffer chamber upper Integrated allows leak-free operation Criterion empty cassettes empty Criterion Criterion precast gels precast Criterion Criterion wire blotter wire Criterion blotter plate Criterion cell Trans-Blot cell Plus Trans-Blot system Turbo Trans-Blot cell SD Trans-Blot Criterion System

system ™ cell Mini-format systems — accommodate small gels The short cm). x 6.7 separation 8.6 to (up distance maximizes the electrical fieldto yield strength (V/cm) rapid separations with moderate resolution. Use these or development, method analysis, rapid for systems Mini-PROTEAN The limited. are volumes sample when system includes the Mini-PROTEAN cell (with a Tetra ® ■ ■ Vertical electrophoresis cells are made in different size formats accommodate to different gels sizes. Deciding which cell use to depends on the requirements for speed, samples of number the (both throughput and resolution, and gels) as well as thevolume sample of available (Table 2.1). precast gels precast cell Turbo ® ® ® Mini-PROTEAN precast gels: precast Mini-PROTEAN cm 8.3 x 0.1 x 6.4 8.6 x 6.7 x 0.1 cm x 0.1 8.6 x 6.7 gels: precast Gel Ready Mini-PROTEAN precast gels precast Mini-PROTEAN Gel Ready Mini Trans-Blot Mini Mini-PROTEAN Tetra Mini-PROTEAN 3 Dodeca 10.0 x 8.0 cm 10.0 Run 1–4 precast or handcast gels in the Mini-PROTEAN cell Tetra and gelsup 12 to in the Mini-PROTEAN Dodeca cell in mini format faster allows assembly clamp Wing setup and leak-free operation Ready Gel empty cassettes empty Gel Ready plates casting Mini-PROTEAN Criterion blotter Criterion Trans-Blot Trans-Blot Trans-Blot SD cell SD Trans-Blot Mini-PROTEAN System

W x L x thickness Handcast Precast Cassette Dimensions (for Handcasting Gels) Handcasting (for Dimensions Cassette Precast Gel Dimensions Gel Precast Electrophoresis Cells Compatible Gel Formats Gel Compatible Advantages Semi-dry transfer Wet/tank transfer Wet/tank

Compatible Transfer Systems Transfer Compatible 14

Table 2.1. Vertical electrophoresis system selection guide. selection system electrophoresis Vertical 2.1. Table Information Sheet, bulletin 1760 bulletin Sheet, Information PROTEAN II xi/XL Cells Product Brochure, bulletin 2710 Criterion Precast Gel System System Gel Precast Criterion Brochure, bulletin 5535 Mini-PROTEAN Cell Tetra Related Literature Related Electrophoresis Guide

TABLE OF CONTENTS 17 Theory and Product Selection Theory Product and Chapter 3: Sample Preparation3: ElectrophoresisChapter for

CHAPTER 3 SamplePreparation for Electrophoresis the involves Sample preparation of solubilization and extraction of free is that sample a protein a total has that and contaminants suitable for concentration protein The quality sample of electrophoresis. the affect can greatly preparation generated. are that data the of quality the of some and guidelines General protein for commonmost methods in provided are preparation sample chapter. this

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TABLE OF CONTENTS 19 Theory and Product Selection Theory Product and

• • • • • • — — — Cell Culture Mammalian

• • — — — — — — — Soft Tissues

• • • — — — — — — Plant Green Material

• — — — — — — — — Seeds

• • • • • — — — — Fungi Yeast, Yeast,

Algae,

• • • • • — — — — Use gentle cell disruption protocols when the sample consists cells of that lyse such easily, as red blood cells or tissue culture cells Use harsher methods, which are based mainly on biological with 2008), (Goldberg mechanical rupture materials that have tough cell walls (for example, plant cells, tissues, and some microbes) When working with a new sample, use at least two different cell disruptionprotocols and compare their efficiencyterms in of yield (by protein assay) and qualitative protein content (by SDS-PAGE) Optimize the power settings mechanical of rupture systems and incubation times for all lysis approaches Mechanical cell lysis usually generates heat; use cooling where required avoid overheating to sample the

■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Cell Disruption The effectiveness a cell of disruption method determines the accessibility intracellular of proteins for extraction and solubilization (Huber et al. 2003). different cell require Different materials biological disruption strategies, which can be divided into two main categories: gentle andharsher methods (Table 3.1). Bacteria

Chapter 3: Sample Preparation3: ElectrophoresisChapter for

cells with glass beads glass with cells blenders, or other motorized devices; this approach is best suited for soft, solid tissues Application of gentle abrasion by vortexing example, Dounce and Potter-Elvehjem homogenizers), homogenizers), Potter-Elvehjem and Dounce example, usually, the mortar is filled with liquid nitrogen and the tissue or cells are ground a fine to powder microorganisms with a mortar and pestle; Homogenization with either a handheld device (for suspension through a small orifice at high pressure high at orifice small a through suspension Breaking cells of solid tissues and of ultrasonic waves ultrasonic of Application of shear forces by forcing a cell to avoidto heating and subjected short to bursts another disruption method, such as sonication as such method, disruption another Disruption of a cell suspension, cooled on ice bacterial cells); this method is usually followed by cells, lyticase for yeast cells, and for containing enzymes that digest the cell wall plant for pectinase and cellulase example, (for disruption method, such as sonication as such method, disruption Suspension of cells in iso-osmotic solutions method is usually followed by another solution to solubilize the ; this membrane; cell the solubilize to solution thawing of cells Suspension of cells in detergent-containing cells swell and burst, releasing cellular contents Freezing in liquid nitrogen and subsequent Description Suspension of cells in hypotonic solution;

Keep the sample preparation workflow as simple as handling sample of number the (increasing possible steps may increase variability) With cell or tissue lysates, include inhibitors minimizeto artifacts generated ; by for required generally not are inhibitors protease plasma serum or samples like Determine the amount protein total of in each sample using a protein assay that is compatible with samples your in chemicals Solubilize proteins in a buffer that is compatible with technique electrophoresis corresponding the Use protein extracts immediately or aliquot them into appropriately sized batches and store them at –80°C avoid freeze-thawto cycles

■ ■ ■ ■ ■ homogenization Glass-bead homogenization Mechanical

Grinding French press

Harsher Methods Sonication

Enzymatic lysis

Detergent lysis Freeze-thaw lysis Technique Gentle Methods Methods Gentle Osmotic lysis

■ ■ ■ ■ ■ General Considerations Due the great to diversity protein of sample types and sources, no single sample preparation method works with all proteins; for any sample, the optimum procedure must be determined empirically. However, the following general sample preparation guidelines should be kept in mind avoid a number to common of pitfalls during sample preparation for protein electrophoresis (Posch et al. 2006): Table Suitability 3.1. of cell disruption methods to various sample types.

Quantitation Cell Disruption Cell Protein SolubilizationProtein Preparation for PAGE Concentration (as needed) (as Concentration as necessary for analysis PAGE. by Contaminant Desalting, Removal, Determine the concentration protein of in a to a finalto sample buffer concentration 1x. of sample Adjust protein by assay. the concentration Dilute the sample in the appropriate sample buffer may modify the protein composition of the sample. the of composition protein modify the may For successful proteins must be well PAGE, solubilized. (which can also help concentrate the sample if needed). Remove interfering substances that can negatively impact Use solubilization solutions that contain chaotropic agents, Different biological materials require different cell disruption different disruption cell require Different materials biological to minimizeto the activity proteases of and other enzymes that that are compatible with the electrophoretic technique used. technique electrophoretic the with compatible are that Use either buffer exchange (desalting) or protein precipitation strategies. Use chemical inhibitors and controlled temperature detergents, reducing agents, buffers, and salts as needed and SDS-PAGE (salts, detergents,SDS-PAGE denaturants, or organic solvents).

Buffer Sample Sample Fig. 3.1. Protein sample preparation workflow. preparation sample workflow. Protein 3.1. Fig.

18

Sample Preparation Workflow Sample Preparation Electrophoresis Guide

TABLE OF CONTENTS 21 Links Micro Bio-Spin 6 and Columns Bio-Spin 6 Micro Kits Lysis MicroRotofor Theory and Product Selection Theory Product and

lysis kits ™ columns, which at a contain mM Tris 10 ™ Buffer exchange — size exclusion chromatography is another effective method for removing salts, detergents, and other contaminants Protein precipitation — the most versatile method to selectively separateproteins from other contaminants consists protein of precipitation trichloroacetic by resolubilization by followed (TCA)/acetone acid in electrophoresis sample buffer. A variety of commercial kits can simplify and standardize from isolation protein for procedures laboratory samplesbiological ■ ■ fuzzy bands and narrowing gel of lanes the toward bottom the of gel. If the ionic strength is very high, no bands will appear in the lower part the of gel vertical (a streak will appear instead) and the dye front will be wavy instead straight. of Deionize any sample with a total ionic strength over 50 mM using columns such as Bio-Spin Micro ■ ■ Products for Sample Preparation Sample for Products successful reproducible and to solution Bio-Rad’s MicroRotofor the is preparation sample Common Solutions for Protein Solubilization Solubilization Protein for Solutions Common Ideally, cell lysis and protein solubilization are carried out inthe sample buffer that is recommended for especially technique, particular electrophoresis the for native electrophoresis.If this is not possible or desirable, dilute the protein solution with concentrated electrophoresis sample buffer final yield to 1x a buffer concentration. Formulasfor various sample buffers are provided in Part II this of guide. Removal ofInterfering Substances Success or failure any of protein analysis depends on sample purity. Interfering substances that can negatively impact include SDS-PAGE salts, detergents, denaturants, or organic solvents (Evans et al. 2009). and/or DNA high samples indicate viscous Highly carbohydrate content, which may also interfere with separations.PAGE In addition, solutions at extreme pH values (for example, fractions from ion exchange most of power separation the diminish chromatography) following the of one Use techniques. electrophoresis methods as needed remove to these contaminants: pH suitable for SDS-PAGE. SDS-PAGE. for suitable pH (Figure 3.3), which(Figure provide 3.3), cell lysis and protein extraction protocols tailored the specific to needsof different sample sources. All four kits are based on the same chaotropic protein solubilization buffer (PSB), which contains nondetergent sulfobetaine 201 and urea, thiourea,(NDSB201) and CHAPS for particularly effective solubilization (Vuillard et al. 1995). Chapter 3: Sample Preparation3: ElectrophoresisChapter for ME drops and proteins reoxidize, reoxidize, proteins and drops ME Buffers and Salts Both pH and ionic strength influence protein , making buffer choice important, especially when native electrophoresis conditions are required. Many proteins are more soluble at higher base therefore, pH; is Tris often included proteins elevate the to pH. However, differ in their solubility at different pH values, so different buffers can extract different sets proteins. of The choice buffer of and pH the of sample preparation solution can strongly influence which proteins show up in a separation. Even in the presence detergents, of some proteins have stringent salt requirements maintain to their solubility, but salt should be present only if it is an absolute requirement. Excess salt samples in SDS-PAGE causes *TCEP is included in Bio-Rad’s XT sample buffers. Although TCEP can be added SDS-PAGE to sample buffer,it must first be neutralized with NaOH; otherwise, it will hydrolyze proteins. drive the equilibrium reaction completion. toward If the concentration b of fuzzy or spurious artifactual bands may result. DTT is less volatile and is altered during the disulfide reduction reaction form to a ring structure from its protein favors equilibrium The chain. straight original reduction, so lowerconcentrations DTT of are needed (higher concentrations are recommended for proteins with large numbers disulfide of bonds). tributylphosphine as such Phosphines (TBP) Tris- and carboxyethylphosphine offer (TCEP)* an alternative to thiols as reducing agents because they can be used at lower concentrations and over a wider pH range than sulfhydryl reductants. the AgentsChaotropic hydrogen disrupt urea as such compounds Chaotropic between and both interactions hydrophobic and bonds within proteins. When used at high concentrations, they destroy secondary protein structure and bring proteins solutioninto that are not otherwise soluble. Urea and substituted ureas like thiourea improve Currently, proteins. hydrophobic of solubilization the best solution for denaturing electrophoresis is a combination 7 M urea of and 2 M thiourea in CHAPS. like detergents appropriate with combination Samples containing urea and thiourea canbe used whenin SDS-PAGE diluted sample with SDS-PAGE buffer. The protein solutionshould not be heated above 37ºC because urea and thiourea get hydrolyzed (to cyanate and thiocyanate, respectively) and modify amino acids on proteins (carbamylation), giving rise to heterogeneity. charge artifactual

SH SH S S S S SH SH Reduction Reduction ME is used in large excess in sample buffers to b ME) and dithiothreitol (DTT) disrupt intramolecular and (DTT) intramolecular disrupt ME) dithiothreitol and ME is volatile, evaporates from solution, and reduces b intermolecular disulfide bonds and are used to achieve achieve to used are and bonds disulfide intermolecular in proteins maintain to and unfolding protein complete their fully reduced states (Figure 3.2). b There exchange. disulfide by bonds disulfide protein disulfides, and thiols between free equilibrium an is so Fig. 3.2. Reduction of proteins with DTT. and to maintain solubility. Nonionic detergents detergents Nonionic solubility. maintain to and are not very effectivesuch X-100 as NP-40and Triton zwitterionic proteins; hydrophobic solubilizing at detergents such as CHAPS and sulfobetaines provide higher or ASB-14) (for example, SB 3-10 membrane integral for especially efficiency, solubilization uses commonly PAGE for preparation Sample proteins. the anionic detergent SDS, which is unparalleled in its ability efficiently to and rapidly solubilize proteins. AgentsReducing 2-mercaptoethanol as such agents reducing Thiol ( Detergents zwitterionic, nonionic, classified as are Detergents hydrophobic disrupt they and cationic, and anionic, interactions between and within proteins (Luche et al. especially proteins, membrane proteins, Some 2003). require detergents for solubilization during isolation If this is not possible or desirable, proteins must be prepared in sample solubilization solutions that typically chaotropic including compounds, of number a contain agents, detergents, reducing agents, buffers, salts, and ampholytes. These are chosen from a small list of compounds that meet the requirements, both electrically and chemically, for compatibility with the electrophoretic technique being used. In these cases, the sample will be dilutedhave to with concentrated electrophoresis sample bufferfinal yield to buffer a 1x concentration.

If protein phosphorylation be isstudied, to include vanadate and fluoride as such inhibitors phosphatase Check the efficacyof cellwall disruptionby microscopy light  all extracts extensively for (20,000 x g remove to any insoluble min material; at 15°C) 15 Lyse samplesLyse using at pH >9 either sodium carbonate as a buffering or Tris agent in the lysis solution (proteases are often least active at basic pH) lysis the to inhibitor chemical protease a Add solid particles may block the pores the of gel Disrupt the sample or place freshly disrupted samples agents denaturing strong containing solutions in suchM urea, as 7–9 2 M thiourea, SDS. or In this 2% environment, enzymatic activity is often negligible Perform cell disruption at low temperatures to diminish enzymatic activity phenylmethylsulfonyl Examples include buffer. best results, use a combination inhibitors of in a cocktail inhibitor protease fluoride (PMSF), aminoethyl-benzene sulfonyl sulfonyl aminoethyl-benzene (PMSF), fluoride ketone chloromethyl tosyl (AEBSF), fluoride (TPCK), etone chloromethyl phenyl tosyl (TLCK), benzamidine, (EDTA), acid ethylenediaminetetraacetic example, (for inhibitors protease peptide and leupeptin, pepstatin, aprotinin, and bestatin). For ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Protein solubilization is the process breaking of interactions involved in protein aggregation, for example, Waals der van bonds, hydrogen bonds, disulfide interactions hydrophobic and interactions, ionic forces, (Rabilloud If these interactions 1996). are not prevented, in resulting precipitate, or aggregate can proteins artifacts or sample loss. For successful proteins PAGE, solubilized. well be must Ideally, cell lysis and protein solubilization are carried out in the sample buffer that is recommended for especially technique, particular electrophoresis the when native electrophoresis is the method choice. of Following cell disruption: Solubilization Protein All cell disruption methods cause the release of (phosphatases, hydrolases compartmentalized glycosidases, and proteases) that can alter the protein composition the of lysates. In experiments where relative amounts protein of be are analyzed, to or in experiments involving downstream immunodetection, the data are only meaningful when the protein composition is preserved. enzymatic Avoid degradation usingby one or a combination the of following techniques:

20 Electrophoresis Guide

TABLE OF CONTENTS Electrophoresis Guide Chapter 3: Sample Preparation for Electrophoresis Theory and Product Selection

The kits generate total protein samples that are Sample Quantitation (Protein Assays) Table 3.2. Bio-Rad protein assay selection guide. ready to be applied to SDS-PAGE, IEF, and 2-D Determine the concentration of protein in a sample Quick Start™ Bradford Bio-Rad DC™ RC DC™ gel electrophoresis. Different sample types have (Berkelman 2008) by using protein assays to: different requirements for effective cell disruption, Method ■■ Ensure that the amount of protein to be separated Bradford • • — — and all four kits combine PSB with other elements to is appropriate for the lane dimensions and Lowry — — • • accommodate these specific needs. visualization method Description One-step determination; Standard Bradford Detergent compatible Reducing agent not to be used with high assay, not to be (DC); Lowry assay detergent ■■ ™ MicroRotofor cell lysis kit (mammal) — for use ■■ Facilitate comparison among similar samples; levels of detergents used with elevated modified to save time compatible (RC DC) with samples from mammalian sources, but it may image-based analysis is simplified when equivalent (>0.025% SDS) levels of detergents also be applied to other animal tissues quantities of proteins have been loaded in the lanes (>0.1% SDS) Standard-Concentration Assay ■■ MicroRotofor cell lysis kit (bacteria) — for use of the gel Sample volume 100 µl 100 µl 100 µl 100 µl with both gram-negative and gram-positive The most commonly used protein assays are Linear range 0.125–1.5 mg/ml 0.125–1.5 mg/ml 0.125–1.5 mg/ml 0.2–1.5 mg/ml Related Literature bacterial cultures colorimetric assays in which the presence of protein Low-Concentration Assay Sample volume 1 ml 800 µl 200 µl 200 µl ■■ MicroRotofor cell lysis kit (plant) — for use with causes a color change that can be measured with Linear range 1.25–25 µg/ml 1.25–25 µg/ml 5–250 µg/ml 5–250 µg/ml MicroRotofor Lysis Kits Flier, soft plant tissues and cultured cells a spectrophotometer (Sapan et al. 1999, Noble and Microplate assay volume 5 µl 10 µl 5 µl ** Minimum incubation 5 min 5 min 15 min 15 min bulletin 5517 ■■ MicroRotofor cell lysis kit (yeast) — for use with Bailey 2009). All protein assays utilize a dilution series Assay wavelength 595 nm 595 nm 650–750 nm 650–750 nm yeast cultures (~60 µl wet cell pellet) of a known protein (usually bovine serum albumin or bovine g-globulin) to create a standard curve from proportional to the amount of bound dye, and thus SmartSpec™ Plus Spectrophotometer which the concentration of the sample is derived (for a Related Literature protocol describing protein quantitation, refer to Part II to the amount (concentration) of protein in the The color change observed in protein assays is of this guide). sample. Compared with other protein assays, monitored and quantitated by a spectrophotometer. the is less susceptible to Bio-Rad’s SmartSpec Plus spectrophotometer Modification of Bio-Rad DC Protein Assays Protein Assay for Use with TABLE CONTENTS OF interference by various chemicals that may be (Figure 3.5) has preprogrammed methods for protein The chemical components of the sample buffer and the Thiols, bulletin 1909 present in protein samples* quantitation and a working wavelength range of amount of protein available for assay dictate the type of 200–800 nm. It can be used for routine applications Colorimetric Proteins Assays, ■■ Lowry (Lowry et al. 1951) — combines the reactions assay that may be used (Table 3.2). bulletin 1069 of cupric ions with peptide bonds under alkaline such as: ■■ Bradford assays (Bradford 1976) — are based on SmartSpec Plus conditions and the oxidation of aromatic protein ■■ Quantitation of proteins via the Bradford, Lowry, Spectrophotometer Fig. 3.3. MicroRotofor cell lysis kit. an absorbance shift of Coomassie (Brilliant) Blue residues. The Lowry method is based on the reaction and BCA assay methods G-250 dye under acid conditions. A redder form of Brochure, bulletin 2826 of Cu+, produced by the peptide-mediated reduction ■■ Quantitation of DNA, RNA, and oligonucleotides For contaminant removal Bio-Rad offers the following the dye is converted into a bluer form upon binding of Cu2+, with Folin-Ciocalteu reagent (a mixture of ■■ Monitoring bacterial culture growth (Figure 3.4): to protein. The increase of absorbance at 595 nm is phosphotungstic acid and phosphomolybdic acid in ■■ Simple kinetic assays ■■ ReadyPrep™ 2-D cleanup kit — uses a modification the Folin-Ciocalteu reaction) ■■ Wavelength scans with peak detection of the traditional TCA protein precipitation protocol. End cap End cap ■■ BCA (bicinchoninic acid, Smith et al. 1985) — reacts The kit offers quantitative protein recovery but Reservoir Reservoir + directly with Cu (generated by peptide-mediated Links also ensures easy and reproducible removal of 3 cm reduction of Cu2+) to produce a purple end product. Links interfering substances 2 cm working bed height 5 cm The reagent is fairly stable under alkaline conditions ■■ Bio-Spin® and Micro Bio-Spin 6 columns — provide 0.8 ml bed volume Sample Buffers 3.7 cm working bed height and can be included in the solution to make Rotofor and Mini rapid salt removal in an easy-to-use spin-column and Reagents the assay a one-step procedure Rotofor Cells format. Accommodating up to 100 µl of sample, 1.2 ml bed volume Luer end fitting with snap-off tip Protein Assay Kits MicroRotofor Cell these columns remove compounds <6 kD; proteins Porous 30 µm Micro Bio-Spin To measure protein concentration in Laemmli buffers, and Cuvettes polyethylene bed Column ™ can be eluted in electrophoresis sample buffer support retains use the reducing agent detergent compatible (RC DC ) MicroRotofor Lysis Kits fine particles protein assay, which is compatible with reducing Disposable Cuvettes for Protein Assays MicroRotofor Cell Lysis Kit Luer end fitting agents and detergents. For more information on protein with snap-off tip (Mammal) quantitation using colorimetric assays, refer to Bio-Rad Quick Start Bradford Bio-Spin Column ReadyPrep 2-D Fig. 3.5. SmartSpec Plus spectrophotometer. Protein Assay Cleanup Kit bulletin 1069. MicroRotofor Cell Lysis Kit (Bacteria) *The Bradford assay is, however, highly sensitive to ionic detergents Features built into the SmartSpec assay methods Bio-Rad Protein Assay like SDS. facilitate data collection and present a complete MicroRotofor Cell Lysis Kit DC Protein Assay (Plant) analysis of assay results. Bio-Rad also offers compatible quartz and UV-transparent plastic cuvettes. RC DC Protein Assay MicroRotofor Cell Lysis Kit (Yeast) SmartSpec Plus Fig. 3.4. Bio-Rad products that can be used for contaminant Spectrophotometer removal. Top, Micro Bio-Spin and Bio-Spin columns; Bottom, Bio-Spin 6 and Micro ReadyPrep 2-D cleanup kit. Bio-Spin 6 Columns

ReadyPrep 2-D Cleanup Kit

22 23 25 Theory and Product Selection Theory Product and

Chapter 4: Reagent Selection and Preparation4: Chapter

This chapter details how to select select to how details chapter This (protein reagents the prepare and required buffers) and gels, standards, applications. various PAGE for buffers and selected gels The types of protein the of size the suit should desired the under investigation, overall the and technique, analysis experiment. the of goals CHAPTER 4 Reagent Selection and Preparation

24 Electrophoresis Guide

TABLE OF CONTENTS Electrophoresis Guide Chapter 4: Reagent Selection and Preparation Theory and Product Selection

Select protein standards that offer: they contain a known amount of protein in each Bio-Rad offers the following prestained and unstained Links General Considerations Related Literature No particular gel type or buffer is useful for all proteins, ■■ Good resolution of the proteins in the size range band, they also allow approximation of protein natural standards (Figure 4.2): and choosing the buffer systems and gel types that of interest concentration. These standards are compatible with ■■ Kaleidoscope standards — individually colored Recombinant Protein offer the highest resolution in the size range of interest Laemmli and neutral pH buffer systems and are an Acrylamide Polymerization — Standards (Markers) ■■ Compatibility with downstream analysis (for proteins to allow instant orientation on gels and blots A Practical Approach, bulletin 1156 may require some experimentation. In selecting excellent choice for use with Stain-Free™ technology example, blotting) ■■ Prestained SDS-PAGE standards — blue colored Precision Plus Protein reagents for PAGE, consider the following: (since they do not contain dye that can interfere with The Little Book of Standards, standards available in low, high, and broad ranges Unstained Standards Protein standards are available as prestained or stain-free detection). See Stain-Free Technology box bulletin 2414 ■■ Protein standards — select protein standards that unstained sets of purified or recombinant proteins. In in Chapter 6 for more details ■■ Unstained SDS-PAGE standards — available in low, Protein Standards Application Precision Plus Protein provide maximum resolution in the size range of general, prestained standards allow easy and direct high, and broad ranges. These standards are blended Guide, bulletin 2998 Prestained Standards interest and that offer compatibility and utility for ■■ Precision Plus Protein prestained standards to give uniform band intensities when stained with Increase Throughput downstream applications such as western blotting visualization of their separation during electrophoresis (All Blue, Dual Color, and Kaleidoscope) — include Precision Plus Protein Coomassie (Brilliant) Blue R-250 or stains with Multiplex Fluorescent Detection, and their subsequent transfer to membranes. Although a proprietary staining technology that provides All Blue Standards ■■ Gel percentage — choose the percentage that offers bulletin 5723 prestained standards can be used for size estimation, batch-to-batch molecular mass consistency and ■■ Polypeptide SDS-PAGE standards — for molecular the best resolution in the range of interest Precision Plus Protein Precision Plus Protein unstained protein standards will provide the most reproducible migration. The ability to visualize these weight determination of peptides and small proteins Dual Xtra Standards—New Protein Dual Color Standards ■■ Handcast vs. precast gels — precast gels offer accurate size determinations. resolved on Tricine gels standards makes them ideal for monitoring protein Standards with an Extended Range greater convenience and superior quality control and separation during gel electrophoresis from 2 to 250 kD, bulletin 5956 Precision Plus Protein Applications and details of Bio-Rad’s protein standards Polyacrylamide Gels Dual Xtra Standards reproducibility than handcast gels; handcast gels are provided in Table 4.1. ■■ provide customized percentages and gradients Precision Plus Protein Dual Xtra standards — Polyacrylamide is stable, chemically inert, electrically Precision Plus Protein Recombinant Standards prestained standards with additional 2 and 5 kD bands neutral, hydrophilic, and transparent for optical Links ■■ Gel format — select mini- or midi-format gels when Kaleidoscope Standards to enable molecular mass estimation below 10 kD throughput is important or sample size is limited; Recombinant standards are engineered to display detection at wavelengths greater than 250 nm. ■■ Precision Plus Protein™ WesternC™ standards — dual Precision Plus Protein select large-format gels for higher resolution. Select specific attributes such as evenly spaced molecular These characteristics make polyacrylamide ideal Natural Standards WesternC Standards weights or affinity tags for easy detection. Bio-Rad’s color, prestained, and broad range protein standards for protein separations because the matrix does not a comb type and gel thickness to accommodate the Prestained recombinant standards are the Precision Plus Protein that enable chemiluminescence detection when probed interact with the solutes and has a low affinity for Precision Protein sample number and volume you are working with SDS-PAGE Standards

TABLE CONTENTS OF standards family and are available as stained or with StrepTactin-HRP conjugates; the protein standard common protein stains (Garfin 2009). StrepTactin-HRP ■■ Buffer system — choose the system that offers the Kaleidoscope Conjugate unstained standards (Figure 4.1). These standards appears directly on a film or CCD image. Additionally best resolution and compatibility with the protein and Polymerization Prestained Standards contain highly purified recombinant proteins with this protein standard has fluorescent properties that application of interest Polyacrylamide gels are prepared by free radical enable detection for fluorescent blots* High Range Prestained molecular masses of 10–250 kD (or 2–250 kD for the polymerization of acylamide and a comonomer SDS-PAGE Standards Protein Standards Dual Xtra standards). Natural Standards cross-linker such as bis-acrylamide. Polymerization Low Range Prestained Protein standards are mixtures of well-characterized or ■■ Precision Plus Protein unstained standards — Natural standards are blended from naturally occurring is initiated by ammonium persulfate (APS) with SDS-PAGE Standards recombinant proteins that are loaded alongside protein include three high-intensity reference bands proteins. Although prestained natural standards are tetramethylethylenediamine (TEMED) acting as a samples in a gel. They are used to monitor separation (25, 50, and 75 kD) and contain a unique affinity effective for monitoring gel separation and efficiency, catalyst (Figure 4.3). Riboflavin (or riboflavin-5'- Broad Range Prestained SDS-PAGE Standards as well as estimate the size and concentration of the Strep-tag, which allows detection and molecular they covalently bind the dye in different amounts and phosphate) may also be used as a source of free proteins separated in a gel. weight determination on western blots. These at different locations. This may produce broader bands radicals, often in combination with TEMED and APS. Unstained SDS-PAGE standards offer absolute molecular weight accuracy than are seen with recombinant standards, making Polymerization speed depends on various factors Standards confirmed by mass spectrometry. Because them less desirable for molecular weight estimations. (monomer and catalyst concentration, temperature, High Range Unstained Table 4.1. Applications of Bio-Rad’s protein standards. SDS-PAGE Standards ™ Precision Plus Protein Standards Prestained Natural Standards MW, kD MW, kD MW, kD MW, kD Low Range Unstained

™ SDS-PAGE Standards Phosphorylase b 97.4 MW, kD MW, kD Myosin 200 ™ —250 Myosin 216 Myosin 210 Bovine serum 66.2 Broad Range Unstained albumin b-Galactosidase 132 Triosephosphate 26.6 —150 isomerase SDS-PAGE Standards b-Galactosidase 125 b-Galactosidase 116.3 Ovalbumin 45 Bovine serum 78 Bovine serum 101 Myoglobin 17.0 Dual Color Dual Kaleidoscope Dual Xtra All Blue WesternC Unstained Range High Range Low RangeBroad Natural Kaleidoscope Polypeptide SDS-PAGE —10 0 albumin albumin Phosphorylase b 97.4 -Lactalbumin 14.4 b Standards Electrophoresis — 75 Carbonic Ovalbumin 56.2 31 Accurate MW estimation • • • • • • — — — — Carbonic 45.7 anhydrase Aprotinin 6.5 anhydrase Bovine serum Coomassie Stains Visualize electrophoresis • • • • • — • • • • Carbonic albumin 66.2 — 50 anhydrase 35.8 chain B, Orientation • • • — • — — — — — Soybean trypsin 32.5 oxidized 3.5 inhibitor Soybean trypsin 29 Soybean trypsin Extended MW range — — • — — — — — — — — 37 inhibitor 21.5 Bacitracin 1.4 inhibitor R-250 Stain Coomassie staining • • • • • • • • • • Lysozyme 21 — 25 Lysozyme Fluorescent staining — — — — — • — — — — 18.4 6.9 Aprotinin Ovalbumin 45 14.4 Coomassie Brilliant Blue — 20 Aprotinin 7.6 Lysozyme Blotting G-250 Stain Monitoring transfer efficiency • • • • • — • • • • — 15 Kaleidoscope Broad range High range Low range Polypeptide SDS-PAGE — 10 prestained prestained SDS-PAGE SDS-PAGE Coomassie staining • • • • • • • • • • standard stained — 5 standard SDS-PAGE standard stained standard stained Immunodetection — — — — • • — — — — standard with Coomassie with Coomassie with Coomassie Fluorescent blots* • • • • • — — — — — — 2 R-250 stain R-250 stain G-250 stain MW = molecular weight. Dual Color Kaleidoscope Dual Xtra All Blue WesternC Unstained * For use with fluorescent blots, not to be confused with fluorescent total blot stains. Precision Plus Protein Fig. 4.2. Bio-Rad’s natural protein standards. prestained standards contain dyes with fluorescent properties. See bulletin 5723 for details on using Fig. 4.1. Precision Plus Protein family of protein standards. precision Plus Protein WesternC standards for fluorescent multiplexing. *Prestained standards are not recommended for use on gels that will be stained with fluorescent dyes.

26 27 29 Links Mini Format 1-D Electrophoresis Systems Ready Gel Precast Gels Gels Precast Mini-PROTEAN Midi Format 1-D Electrophoresis Systems Gels Precast Criterion Theory and Product Selection Theory Product and

15 µl 15 15 µl 15 20 µl 45 µl 30 µl 30 µl 800 µl Well Volume 7 cm IPG strip 30 µl and 50 µl 11 cm IPGstrip 11

Comb Thickness, mm 1.0 12 15 18 10 26 IPG 8+1 12+2

IPG+1 Prep+2 Number of Wells

) ® and ® ) ™ Cast the gel Prepare the sample (sample buffer) Fill the electrode reservoirs (running buffer) Chapter 4: Reagent Selection and Preparation4: Chapter ■ ■ ■ ■ ■ ■ Buffer Systems and Gel Chemistries (Criterion Most applications common PAGE utilize discontinuous buffer systems (Niepmann where 2007), two ions moving a mobility form differing electrophoretic in boundary when a voltage is applied (see Chapter 2). Proteins have an intermediate mobility, making them stack, or concentrate, a narrow into zone at the moves zone that As electrophoresis. of beginning through the gel, the sieving effect the of gel matrix causes proteins different of molecular weights move to at different Varying rates (see Figure the types 2.2). ionsof used in the buffers changes the separation characteristics and stability 4.3 the of gel. Table summarizes the various types gel of and buffer Format (Size and Comb Type) The size format the of gel used depends on the electrophoresis cell selected Precast (see Chapter 2). midi-format and mini- Bio-Rad’s for available are gels electrophoresis systems, and handcasting accessories are available fit to all Bio-Rad electrophoresis cells. Additional parameters consider to include the number wellsof and gel thickness, which depend on the number and volume samples of create analyze. to To sample wells in a gel, a comb is placedthe into top theof gel prior polymerization. to When the comb is removed, a series sample of wells is left behind. The number and size these of wells dictate how many samples and what volume may beloaded (Table 4.2). The thickness the of gelalso plays a role in determining the sample volume that can be loaded. A variety of comb types are available for handcasting; refer to information. more for www.bio-rad.com Table 4.2. Comb types available for Bio-Rad precast polyacrylamide gels. The pH and ionic composition the of buffer system determine the power requirements and heavily influence the separation characteristics a polyacrylamide of gel. Buffer systems include the buffers used to: available. systems Mini-Format Gels Gel(Ready Mini-PROTEAN

Gels Midi-Format

. Use single-percentage gels separate to bands thatare close in molecular weight. Since optimum separation occurs in the lower half the of gel, choose a percentage in which your protein interest of migrates the lower to half the of gel Use gradient gels separate to samples containing a broad range molecular of weights. Gradient gels low-molecular and high- both of resolution allow weight bands on the same gel. The larger pore size the the of toward top gel permits resolution larger of molecules, while pore sizes that decrease toward the bottom the of gel restrict excessive separation of molecules small For new or unknown samples, use a broad gradient, for a globalsuch as evaluation 4–20% or 8–16%, theof sample. Then using move to an appropriate single-percentage gel once a particular size range of proteins has beenidentified ■ ■ ■ ■ ■ ■ Precast vs. Handcast vs. Precast Precast gels are ready use to and offer greater convenience, more stringent quality control, and higher gels precast Many gels. handcast than reproducibility also provide months, a shelf allowing life 12 up of to gels be storedto and used as needed (this is not possible with handcast gels, as they degrade within a few days). Handcast gels, on the other hand, must be prepared acrylamide from bisacrylamide and solutions; monomer the component solutions are prepared, mixed and thentogether, poured between two glass plates polymerizeto (see Part II this of guide for a detailed Because acrylamide bisacrylamide and are protocol). neurotoxins when in solution, care must be taken to avoid direct contact with the solutions and clean to up any spills. In addition, the casting process requires hours complete, to is not as controlled as it is gel by manufacturers, and contributes more to irregularities and lessreproducibility with handcast gels. customized of benefit offers the handcasting Although percentages,chemistries, and gradients, precast gels are sized fit to specific electrophoresis cells and are available in a range chemistries, of formulations, comb types, and thicknesses. differfrom gels Precast their handcast counterparts in that they are cast with a single buffer throughout. Bio-Rad’s precast gels also(Table 4.3) do not contain SDS and can be used for native or denaturing For a complete and PAGE. current list available of precast gels, visit the Bio-Rad website at www.bio-rad.com 66 45 31 66 45 31 75 50 37 25 20 15 10 6.5 75 50 37 25 20 15 10 6.5 200 116 200 116 Any kD Any kD 250 150 100 97.4 21.5 14.4 Any kD Any kD 250 150 100 97.4 21.5 14.4 66 45 31 66 45 31 6.5 6.5 75 50 37 25 20 15 10 4–20% 4–20% 200 116 10 75 50 37 25 20 15 4–20% 4–20% 200 116 97.4 21.5 14.4 97.4 21.5 14.4 250 150 100 250 150 100 Unstained Unstained

66 45 31 66 45 31 6.5 6.5 4–15% 4–15% 75 50 37 25 20 15 10 4–15% 4–15% 75 50 37 25 20 15 10 200 116 200 116 21.5 14.4 97.4 97.4 21.5 14.4 250 150 100 250 150 100 45 31 12% 12% 66 45 31 12% 12% 66 75 50 37 25 20 15 20 37 25 15 75 50 200 116 200 116 21.5 14.4 97.4 97.4 21.5 14.4 250 150 100 250 150 100 Broad Range Unstained Broad Broad Range Unstained Broad n Plus Protei Precision n Plus Protei Precision Mini-PROTEAN TGX Mini-PROTEAN TGX 10% 10% 31 66 45 10% 10% 66 45 31 25 20 75 50 37 50 25 20 75 200 116 37 200 116 21.5 97.4 97.4 21.5 250 150 100 250 150 100 45 31 66 7.5% 7.5% 66 45 31 7.5% 7.5% 50 37 50 37 75 75 200 116 200 116 250 150 100 250 150 100 97.4 97.4 Fig. 4.4. Examples of migration charts. migration Examples of 4.4. Fig. Gels can be madewith a single, continuous percentage throughout the gel (single-percentage or they gels), can be cast with a gradient through the gel %T of (gradient gel Typical gels). compositions and are between 7.5% for single-percentage20% gels, and typical gradients and Use protein 10–20%. migration chartsare 4–15% and tables select to the gel type that offers optimum resolution your of sample (Figure 4.4):

CH CH C x 100 N O H CH CH x100

O O CH NH C C NH CH CH CH Acrylamide monomer C CH CH N O O O H Cross-link NH C C NH CH CH Total Total volume, ml Polyacrylamide CH CH CH CH O O C O g acrylamide + g cross-linkerg + acrylamide g g cross-linker g g acrylamide g + cross-linker NH C C NH CH CH CH NH %T = %C = CH CH O O CH NH NH C CH CH C 19:1, 29:1, or 37.5:1 on acrylamide/bis solutions or 37.5:1 29:1, 19:1, represent cross-linker and 2.7% 3.3%, ratios 5%, of (the most common cross-linker concentrations for protein separations). The practical ranges for monomer concentration are stock solutions 30–40%, of with different ratios of designations The cross-linker. acrylamide to monomer Percentage Polyacrylamide gels are characterized two by parameters: in monomer total concentration (%T, ml) andg/100 weight percentage cross-linker of (%C). By varying these two parameters, the pore size the of gel can be optimized yield to the best separation and resolution for the indicates proteins interest. of %T the relative pore size the of resulting polyacrylamide gel; a higher refers a larger to %T ratio polymer-to-water and smaller average pore sizes. Fig. 4.3. Polymerization of acrylamide and monomers of Polymerization 4.3. Fig. bisacrylamide. and purity reagents) of and must be carefully controlled because it generates heat and may lead to nonuniform pore structures if it is rapid. too NH cross-linking monomer cross-linking N,N’-Methylenebisacrylamide C CH O

SDS-PAGE Standards SDS-PAGE Broad Range Unstained Unstained Range Broad Unstained Standards Unstained Links Precision Plus Protein

28 Electrophoresis Guide

TABLE OF CONTENTS 31 Related Literature Related Mini-PROTEAN TGX Precast Sheet, Information Product Gels bulletin 5871 Mini-PROTEAN TGX Precast Gel: A Gel for SDS-PAGE with Improved Stability — Comparison with Standard Laemmli Gels, bulletin 5910 Mini-PROTEAN TGX Precast Gel: A Versatile and Robust Laemmli-Like Precast Gel for 5911 bulletin SDS-PAGE, Ready Gel Mini-PROTEAN to TGX Precast Gels Catalog Number 5932 bulletin Chart, Conversion PrecastNuPAGE Bis-Tris Gels (MOPS Buffer) to Mini-PROTEAN TGX Precast Gels Catalog Number 5934 bulletin Chart, Conversion Criterion XT Precast Gels Product 2911 bulletin Sheet, Information Criterion TGX Stain-Free Information Product Gels Precast Sheet, bulletin 5974 Links Mini Format 1-D Electrophoresis Systems Mini-PROTEAN TGX Gels Precast Midi Format 1-D Electrophoresis Systems Criterion TGX Stain-Free Gels Precast Criterion XT Bis-Tris Gels Precast Trans-Blot Turbo System Transfer Systems Imaging System MP ChemiDoc Gel Doc EZ Imaging System Stains Coomassie Coomassie Brilliant Blue R-250 Stain Coomassie Brilliant Blue G-250 Stain Reagents Buffers and Sample BuffersRunning Reagents and Theory and Product Selection Theory Product and XT ™ ME and DTT Chapter 4: Reagent Selection and Preparation4: Chapter running buffer produces different migration patterns: patterns: bufferrunning different migration produces MES bufferis used for small proteins, and MOPS buffer is used for mid-sized proteins. gels (for example,Precast Criterion Bis-Tris IEF Isoelectric focusing (IEF) separates proteins their by net charge rather than molecular weight. IEF gels molecules amphoteric ampholytes, with cast are that generate a pH gradient across the gels. Proteins migrate their to pI, the pH at which the protein has no net charge. Since IEF gels contain no denaturing agents, IEF is performed under native conditions. Zymogram Zymogram gels contain either gelatin or casein, which are substrates for various proteases. Samples are run are and conditions nonreducing but under denaturing then allowed renature to and consume the substrate. Samples with proteolytic activity can be visualized as clear bands against a blue background after using Coomassie (Brilliant) stain. Blue R-250 Tris-Acetate Tris-Acetate This discontinuous buffer system uses acetate as the leading ion and as the Tricine trailing ion and is ideally suited large of for SDS-PAGE proteins. Tris-acetate gels can be used for both SDS- and native PAGE. gels, they offerLike extended Bis-Tris shelf life and room temperature storage. Because their of lower pH, these gels offer better stability than gels Tris-HCl and are best suited for peptide sequencing and mass applications. spectrometry Tris-Tricine One the of drawbacks using to SDS in a separation system is that excess SDS runs as a large front at the low molecular weight end the of separation. Smaller polypeptides do not separate from this front and, therefore, do not resolve discrete into bands. Replacing the glycine in the Laemmlirunning buffer with Tricine yields a system that separates the small SDS- polypeptides from the broad band SDS of micelles that forms behind the leading-ion front. Proteins as small as 1–5 kD can be separated in these gels. Bis-Tris gels) offer extendedBis-Tris shelf life (compared to gels) andTris-HCl room temperature storage. These gels are popular because their of stability but they require special buffers, and the gel patterns cannot be compared those gels. to Tris-HCl of Common reducing agents such as b are not ionized at the relatively low pH Bis-Tris of gels and so do not enter the gel and migrate with the proteins. Alternative reducing agents are, gelstherefore, maintain to used with a Bis-Tris reducing environment and prevent protein reoxidation electrophoresis. during

™

tended X EZ or ™ lycine e lycine G ris- T ( ™ gels are Laemmli-like, Laemmli-like, gels are ™ system), and theysystem), do not require ™ MP imaging systems, eliminating staining/ eliminating systems, imaging MP Turbo ™ ®

ChemiDoc shelf life) precast gels, modified Laemmli gels with a 12 to life shelf extends that proprietary modification months and allows gels be run to at higher voltages without producing excess heat. The formulation TGX yields run times as short min and Laemmli-like as 15 separation patterns with exceptionally straight lanes and sharp bands. gels TGX offer excellent staining quality and transfer efficiency(with transfer times as short as min for tank blotting15 and as short as 3 min with the Trans-Blot destaining steps for completion protein of separation, visualization, and analysis min in (see 25 Stain-Free special, expensive buffers. gels, Like Tris-HCl TGX gels use a discontinuous buffer system, with glycinate as the trailing ion, and are, therefore, compatible with conventional Laemmli and buffers. Tris/glycine/SDS These are the best choice when long shelf life is needed and traditional Laemmli separation patterns are desired. Stain-Free TGX Bio-RAD’S Bis-Tris Bis-Tris These systems employ chloride as the leading ion and MES or MOPS as the trailing ion. The common cation buffer. Theis formed gels are from prepared Bis-Tris enhance to at pH 6.4 gel stability. Running the same gels with eitherBis-Tris MES or MOPS denaturing Laemmli (Tris-HCl) (Tris-HCl) Laemmli The Laemmli system has been the standard system for SDS- applications and native PAGE for many years. Many researchers gels use Tris-HCl because the reagents are inexpensive and readily available; precast gels are also readily available in a wide variety gelof percentages. This discontinuous buffer system relies on the stacking effect a moving of boundary formed between the leading ion (chloride) and the trailing ion Tris (glycinate). buffer is the common cation. gels Tris-HCl can be used in either denaturing mode SDS-PAGE (using Laemmli sample buffer and running Tris/glycine/SDS buffer) or modein (using native native PAGE sample and running buffers without denaturants or SDS). resolvingTris-HCl gels are prepared at pH 8.6–8.8. thisAt basic pH, polyacrylamide slowly hydrolyzes to separation. compromise can which polyacrylic acid, For this reason, gels Tris-HCl have a relatively short shelf life. In addition, the gel pH can rise during pH 9.5 to a run, causing proteins undergo to deamination and complicate and resolution diminish may This alkylation. analysis. postelectrophoresis TGX developed has Bio-Rad extended shelf life gels that allow rapid fluorescent detection proteins of with the Gel Doc Technology in box Chapter 6 for more details).

• • • • • • — — — — — — — — . Handcast

• • • • ­• • • • • • • • • • Gels Criterion

• • • • • • • • • — — — — — Precast (Format) (Ready Gel) (Ready Gel) (Ready Gel) (Ready Gel) (Mini-PROTEAN) (Mini-PROTEAN) (Mini-PROTEAN) (Mini-PROTEAN) (Mini-PROTEAN) Mini-PROTEAN*

Running Tris/glycine/SDS

IEF anode buffers Tris/glycine/SDS IEF cathode and Tris/glycine/SDS

Tris/glycine/SDS Tris/glycine Tris/glycine Tris/glycine Tris/Tricine/SDS

XT MOPS or Tris/glycine Tris/glycine

XT MES XT Tricine or Tris/Tricine/SDS Tris/glycine/SDS Buffers

Sample Laemmli Zymogram 50% glycerol Laemmli Laemmli Native Native Native Tricine XT Native Native XT or Tricine Laemmli

ampholytes, allow

®

Easy prepare, to reagents inexpensive switching between precast and handcast gels and need compare to results and readily available; best choice when separation by protein pl; contain no denaturing agents, so IEF is performed conditions native under Cast with gelatin or casein, which acts as a substrate for proteases that are separated in the gel under nondenaturing Laemmli-like gels with trihalo compounds without detection fluorescence rapid for best choice when long shelf life is needed Laemmli separation traditional and Laemmli-like extended shelf life gels; patterns are desired weight proteins and protein complexes Optimized for separating peptide and Laemmli-like extended shelf life gels; best choice when long shelf life is needed and Laemmli patterns separation traditional are desired Laemmli-like gels with trihalo compounds staining without detection Offer best separation of high molecular proteins with molecular weight <1,000 Cast with Bio-Lyte for rapid fluorescence detection without detection fluorescence rapid for Laemmli-like extended shelf life gels with fluorescence rapid for compounds trihalo detection without staining staining without detection Offer longest shelf life, but reagents spectrometry mass or sequencing Retention of native protein structure, resolution of proteins with similar weight molecular may be costly trihalo compounds for rapid fluorescence rapid for compounds trihalo Offer best resolution of high molecular weight proteins; useful in peptide Laemmli-like extended shelf life gels with

Selection CriteriaSelection

™

™

™

TheMini-PROTEAN gel running systems are compatible with both Ready Gels and Mini-PROTEAN gels. For gel typesMini-PROTEAN available compatible in a format, the table indicates whether they are available as Ready Gels or Mini-PROTEAN gels. SDS-PAGE Tris-HCI, pH 8.6 TGX

*  Protease Detection Zymogram conditions staining Stain-Free TGX

Stain-Free pH 7.0 Analysis Peptide Tris-Tricine TGX Stain-Free Tris-acetate, IEF IEF staining TGX Stain-Free pH 6.4 Bis-Tris, applications PAGE Native Tris-HCI, pH 8.6

Tris-acetate, pH 7.0 Table 4.3. Gel and buffer chemistries for PAGE. For a current list of precast gels available from Bio-Rad, visit www.bio-rad.com

Gel Type Gel

30 Electrophoresis Guide

TABLE OF CONTENTS Electrophoresis Guide Chapter 4: Reagent Selection and Preparation Theory and Product Selection

Products for Handcasting Gels mixed in a gradient former before being introduced into The following products are available to facilitate the gel cassette. Typically, two solutions are prepared: handcasting gels. For detailed handcasting protocols, the light solution (equivalent to the lowest %T in the refer to Part II of this guide. range to be poured) and a heavy solution (equivalent to the maximum %T to be poured). The most common Premade Buffers and Reagents Related Literature gradient gel contains 4–20% acrylamide; however, the Electrophoresis buffers and reagents are available range of acrylamide concentrations should be chosen as individual reagents or as premixed gel-casting, on the basis of the size of the proteins being separated. Ready-to-Run Buffers and sample, and running buffers. Use of commercially Solutions Brochure, prepared, premixed buffers, which are made with bulletin 2317 Two gradient formers are available for PAGE systems. electrophoresis-purity reagents and are quality Depending on the gel format, prepare either a single controlled for reproducible results, saves time but gel using the gradient former or couple the gradient also maximizes reproducibility, prevents potential former with a multi-casting chamber to prepare up mistakes in buffer concentration, and standardizes to 12 gels simultaneously (Figure 4.6). electrophoresis runs. There are no reagents to weigh ■■ Use the Model 485 gradient former to cast a or filter; just dilute with distilled or deionized water. minimum of 4 mini-format gels at a time using the AnyGel™ Stands Mini-PROTEAN 3 multi-casting chamber, or to cast AnyGel stands (Figure 4.5) provide stabilization and a single, large-format (PROTEAN II or PROTEAN access to gels for casting and sample loading. The Plus) gel clamping mechanism secures gel cassettes vertically ■■ Use the Model 495 gradient former to prepare 4–12 without excess pressure. They are available in two large-format gels (PROTEAN II and PROTEAN Plus) sizes, single- and six-row. using the multi-gel casting chambers TABLE CONTENTS OF

Fig. 4.5. AnyGel stands.

Multi-Casting Chambers Multi-casting chambers are used to cast multiple gels of various thicknesses simultaneously. Acrylic blocks Links act as space fillers when fewer than the maximum number of gels are cast. These chambers work in concert with the gradient formers through a bottom AnyGel Stand filling port to ensure reproducibility. Multi-casting Fig. 4.6. Multi-casting chambers and gradient formers. Polyacrylamide Gel chambers are available for casting gels for the Reagents Mini-PROTEAN, PROTEAN® II, and PROTEAN Premixed Casting Buffers Plus systems. Gradient Formers Mini-PROTEAN Tetra Handcast Systems Gradient gels have a gradient of acrylamide concentration that increases from top to bottom. To PROTEAN Plus create this gradient, the acrylamide solutions must be Multi-Casting Chamber

PROTEAN Plus Hinged Spacer Plates and Combs

Model 485 Gradient Former

Model 495 Gradient Former

32 33 35 Theory and Product Selection Theory Product and Chapter 5: Performing Electrophoresis Performing 5: Chapter

In this phase of the workflow, workflow, the of phase this In is system electrophoresis the samples loaded, assembled, are by is initiated and electrophoresis supply. power the programming Select running conditions that resolution while optimum provide the of temperature the maintaining during separation. system CHAPTER 5 Performing Electrophoresis

34 Electrophoresis Guide

TABLE OF CONTENTS 37 Links Power Supplies High-Current HC PowerPac Power Supply PowerPac Universal Power Supply System Drying GelAir Theory and Product Selection Theory Product and drying system, which allows allows which drying system, ™ Chapter 5: Performing Electrophoresis Performing 5: Chapter Increaserun times for gradient gels and decrease them as needed for low molecular weight proteins minof a run,~10 allow theFor the first sample to stack gelusing V/cm length. a field Then strength of 5–10 in recommended maximum voltage the with continue the instruction manual the of electrophoresis system If using multiple cells and constant voltage, use the same voltage for multiple cells as you would for one cell. The current drawn the by power supply doubles with two — compared one to — cells. Set the current limit high enough permit to this additive function. Also be sure use to a power supply that can current additive this accommodate ■ ■ ■ ■ ■ ■ gels dry to between two sheets cellophane of within 60 min. This yields publication-quality clear, gels ideal densitometry. for General Guidelines for Running Conditions Running for Guidelines General settings different power require cells Electrophoresis with different buffer systems. Thevalues presented are guidelines — conditions should be optimized for each application. In every case, run the gel until the dye front reaches the bottom the of gel. Use external cooling duringlong, unsupervised runs. and uniform more yield often runs Temperature-controlled results. reproducible For best results: Storage Disassembly and Gel Remove the gel cassette and open it according to the manufacturer’s instructions. Before handling the gel, wet your gloves with water or buffer keep to the gel from sticking and minimize to the risk tearing. of Sometimes it is also helpful lift to one edge the of gel with a spatula. Stain, or blot, process the gel as soon as possible maintainto the resolution achieved during electrophoresis and keep to the gel from drying out (see Chapters 6 and For long-term 7). storage, either dry stained glycerol gels in solution a 10% (storage at or with the4°C) GelAir HC and PowerPac Universal ™ In continuous buffer systems (for example, those used for blotting or DNA separation), resistance by caused temperature increasing with declines heating Joule In discontinuous systems, such as the Ornstein-Davis and(native) Laemmli systems, (SDS-PAGE) resistance also changes as discontinuous buffer ion fronts move through resistance the gel; in SDS-PAGE, increases as the run progresses. Depending on the buffer and which electrical parameter is held constant, Joule heating the of gel may increase or decrease over the period the of run ■ ■ power supplies also have an automatic crossover capability that allows the powersupply switch to over a variableto parameter if a set output limit is reached. This prevents damage the electrophoresis to cell. The resistance, does not remain however, constant during a run: ■ ■ If the current is held constant during a run, the voltage, and consequentlypower, the heat the of gel chamber increase during the run. As a rule, constant current conditions result in shorter but hotter runs than runs. constant voltage Power Constant Under Separations Holding the power constant minimizes the risk overheating. of Selecting Power Supply Settings Power supplies that are used for electrophoresis hold or current, voltage, (either constant parameter one power). The PowerPac Voltage Constant Under Separations If the voltage is held constant throughout a separation, the current and power (heat) decrease as the resistance increases. This leads increased to run times, which allow the proteins more time diffuse. to But this appears be offset to the by temperature-dependent increase in diffusion the of rate constant current mode. Separations using constant voltage are often preferred because a single voltage that is independent the of number gels of being run is specifiedfor each gel type. Constant CurrentSeparations Under Heat = P/4.18 cal/sec = P/4.18 Heat Alterations buffer to composition; that is, the addition SDS of or changes in ion concentration due the addition to acid of or base adjust to the pH a buffer of Gel pH, ionic strength, and percentage acrylamide of Number gels of (current increases as the number gelsof increases) Volume buffer of (current increases when increases) volume when increases (current temperature Transfer temperature increases) higher demands length gel (increasing length Gel voltage settings increase to field strength accordingly) Gel thickness (increasing gel width or thickness at identical gel length leads higher to current; voltage unchanged) kept be must ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Other Factors AffectingOther Factors Electrophoresis The following variables also change the resistance theof system and, therefore, affect separation efficiency readings: voltage and current and Understanding the relationships between power, voltage, current, resistance, and heat is central to efficiency the influence that factors understanding the and efficacyof electrophoresis. The optimum condition runis to at the highest electric field strength possible within the heat dissipation capabilities the of system. During an electrophoretic separation usingthe Ornstein- Davis and Laemmli systems, the running buffer warms as a result Joule of heating. The increase in temperature may lead inconsistent to field strength and separation and may cause the buffer lose to its buffering capacity or the gel melt to or become distorted. Under normal running conditions, the running buffer absorbs most of the heat that is during generated. extended However, runs or high-power conditions, active buffer cooling is required prevent to uncontrolled temperature increases. thatoccurs dependson the conductivity the of buffer used, the magnitude the of applied field, andtotal the resistance withinthe system. The heat generated is proportional the power to (P): consumed /R 2 R = V 2 E = V/d P = VI = I I = V/R or V = IR or R = V/I V = IR or or I = V/R

Most vertical electrophoresis chambers are operated at a fieldfor 1 mm thick10–20V/cm strength of polyacrylamide gels. Heating Joule The electric that field can V/cm) strengthbe in (E, generated between the electrodes is limited the by heat This electrophoresis. during produced inevitably is that Joule heating can lead band to distortion, increased diffusion, and protein denaturation when not efficiently removed from the system. The amount Joule of heating The strength the of electric fieldapplied E (V/cm) between the two electrodes is an important parameter in electrophoresis, because it exerts a force on electrically charged objects like proteins and determines their migration (where rate d is the distance in cm): Useful Equations Useful separations,In PAGE the gel containing the protein sample is placed in the electrophoresis chamber between two electrodes. The driving force behind the separation is the voltage in volts) applied (V, across the electrodes. This leads a current to flow (I, in amperes) through the gel, which has an intrinsic resistance (R, in law describes Ohm’s ohms). the mutual dependence of these three parameters: The applied voltage and current are determined the by user and the power supply settings; the resistance is inherent in the system and is determined the by ionic strength the of buffer, the conductivity the of gel, and factors. other in watts) consumedThe an by power electrical (P, current element is equal the product to the of voltage current: and Running Conditions Conditions Running allow supplies power (DC) current direct Regulated control over every electrophoresis mode (constant voltage, current, The or power). choice which of electrical parameter control to is usually a matter preference. of System Setup System System setup involves placing the gels in the tank, filling the tank with running buffer, loading the samples and proteinstandards, and programming the power Followsupply. the instructions for system setup in the instruction manuals for the system you are using. General procedures and tips are provided in Part II guide. this of

36 Electrophoresis Guide

TABLE OF CONTENTS 39 Theory and Product Selection Theory Product and

Chapter 6: Protein Detection and Analysis

Following electrophoresis, protein protein electrophoresis, Following visualized can be patterns band and qualitative to subjected and most Since analysis. quantitative with a gel in seen be cannot proteins visualization protein eye, naked the of use through achieved usually is stained, is gel the Once stains. protein analyzed and imaged can be it and instruments imaging using software. accompanying CHAPTER 6 Protein Detection and Analysis

38 Electrophoresis Guide

TABLE OF CONTENTS Electrophoresis Guide Chapter 6: Protein Detection and Analysis Theory and Product Selection

Table 6.1. Bio-Rad Gel stain selection guide. Related Literature Protein Stains In comparison to Coomassie or In many cases, the choice of staining technique techniques, however, fluorescent dyes are more Sensitivity Detection Total Protein Stain (Lower Limit) Time Comments Method depends on the availability of imaging equipment. expensive and require either a CCD (charge-coupled Rapid Validation of Purified Coomassie Stains Colorimetric However, a protein staining technique should offer the device) camera or fluorescence scanner for gel Proteins Using Criterion Stain imaging. For these reasons, fluorescent stains are Bio-Safe Coomassie G-250 8–28 ng 1–2.5 hr Nonhazardous staining in aqueous solution; Free Gels, bulletin 6001 following features (Miller et al. 2006): premixed, mass spectrometry compatible

■■ often used in proteomics applications and on 2-D Sensitivity and Protein- High sensitivity and reproducibility Coomassie Brilliant Blue R-250 36–47 ng 2.5 hr Simple and consistent; mass spectrometry gels, where the relative quantitation of proteins in compatible; requires destaining with to-Protein Consistency of ■■ Wide linear dynamic range complex mixtures is performed over several orders of Flamingo Fluorescent Gel Stain Silver Stains Colorimetric ■■ Compared to Other Fluorescent Compatibility with downstream technologies abundance and protein identity is determined using Dodeca™ silver stain kit 0.25–0.5 ng 3 hr Simple, robust; mass spectrometry compatible; Stains, bulletin 5705 such as protein extraction and assay, blotting, in-gel proteolytic digestion and mass spectrometry. ideal for use with Dodeca stainers (Sinha et al. 2001) or mass spectrometry Examples include Flamingo™ and Oriole™ fluorescent Silver Stain Plus™ kit 0.6–1.2 ng 1.5 hr Simple, robust; mass spectrometry compatible Comparison of SYPRO Ruby (Gottlieb and Chavko 1987) and Flamingo Fluorescent ■■ Robust, fast, and uncomplicated protocol gel stains Silver stain (Merril et al. 1981) 0.6–1.2 ng 2 hr Stains , lipoproteins, lipopolysaccharides, Gel Stains With Respect ■■ Staining protocols usually involve the following three Silver stains — offer the highest sensitivity, but nucleic acids to Compatibility With Mass with a low linear dynamic range (Merril et al. 1981, Spectrometry, bulletin 5754 steps (protocols are available in Part II of this guide): Fluorescent Stains Fluorescent Rabilloud et al. 1994). Often, these protocols Oriole fluorescent gel stain* 0.5–1 ng 1.5 hr Rapid protocol, requires no destaining, mass spectrometry ■■ Protein fixation, usually in acidic methanol or Oriole Fluorescent Gel are time-consuming, complex, and do not offer compatible; compatible only with UV excitation Stain: Characterization and (a few staining protocols already contain acid or sufficient reproducibility for quantitative analysis. Flamingo fluorescent gel stain 0.25–0.5 ng 5 hr High sensitivity; broad dynamic range; simple protocol requires no destaining; Comparison with SYPRO Ruby alcohols for protein fixation and so do not require mass spectrometry compatible; excellent for laser-based scanners Gel Stain, bulletin 5921 In addition, their compatibility with mass this separate step) spectrometry for protein identification purposes is SYPRO Ruby protein gel stain 1–10 ng 3 hr Fluorescent protein stain; simple, robust protocol; Bio-Safe Coomassie Stain broad dynamic range; mass spectrometry compatible ■■ Exposure to dye solution lower than that of Coomassie stains and fluorescent Brochure, bulletin 2423 Negative Stains Colorimetric ■■ dyes (Yan et al. 2000) Washing to remove excess dye (destaining) Zinc stain 6–12 ng 15 min High-contrast results; simple, fast, and reversible; compatible Flamingo Fluorescent Gel Stain ■■ Negative stains — rapid negative stains require only with elution or blotting as well as mass spectrometry Product Information Sheet, Total Protein Stains (Fernandez-Patron et al. 1992) bulletin 5346 Total protein stains allow visualization of the protein ~15 min for high-sensitivity staining, where protein TABLE CONTENTS OF bands appear as clear areas in a white background. Copper stain 6–12 ng 10 min Single reagent; simple, fast, and reversible; compatible with separation pattern in the gel. Table 6.1 compares the elution or blotting as well as mass spectrometry (Lee et al. 1987) Zinc and copper stains do not require gel fixation, advantages and disadvantages of several total * Do not use Oriole gel stain with native gels. protein staining techniques. ensuring that proteins are not altered or denatured. Links Negative stains can be used as a quality check ■■ Coomassie stains — the most popular anionic before transfer to a western blot or analysis by mass Stain-Free™ Technology protein dye, Coomassie (Brilliant) Blue stains spectrometry, although they are not the best choice Criterion Precast Gels almost all proteins with good quantitative linearity when quantitative information is desired Bio-Rad’s stain-free technology allows direct Benefits of stain-free technology include: at medium sensitivity. There are two variants of visualization, analysis, and documentation ■■ Elimination of staining and destaining steps for Criterion TGX Stain-Free ■■ Stain-free technology — a haloalkane compound Coomassie (Brilliant) Blue: R-250 (R for reddish), of protein samples in PAGE gels without staining, Precast Gels in Bio-Rad’s Criterion™, Criterion™ TGX, and Mini- faster results which offers shorter staining times, and G-250 (G destaining, or gel drying. ® ™ ■■ Mini-PROTEAN TGX PROTEAN TGX Stain-Free gels covalently binds to Automated gel imaging and analysis for greenish), which is available in more sensitive ™ Stain-Free Gels residues of proteins when activated with The system comprises the Gel Doc EZ and ■■ No background variability within a gel or and environmentally friendly formulations (Simpson ™ ™ UV light. This allows protein detection (with a stain- ChemiDoc MP imagers, Image Lab software, between gels (as is often seen with standard Coomassie Stains 2010, Neuhoff et al. 1988). Coomassie dyes are free compatible imager) in a gel both before and and precast gels that include unique trihalo also the favorite stains for mass spectrometry and Coomassie staining) Bio-Safe Coomassie Stain after transfer, as well as total protein detection on a compounds that allow rapid fluorescent detection of protein identification. Bio-Safe™ Coomassie stain is a ■■ Visualization of transferred (blotted) proteins Links blot when using PVDF membranes (see Stain-Free™ proteins — without staining. The trihalo compounds Coomassie Brilliant nonhazardous formulation of Coomassie Blue G-250 on low-fluorescence PVDF membranes Technology box) react with tryptophan residues in a UV-induced Blue R-250 Stain that requires only water for rinsing and destaining. reaction to produce fluorescence, which can be ■■ Reduced organic waste by eliminating the SYPRO Ruby Protein It offers a sensitivity that is better than conventional Specific Protein Stains Gel Stain Coomassie Brilliant detected by the imager either within gels or on low- use of and methanol in staining Coomassie R-250 formulations and equivalent to Specific protein stains are used to visualize specific Blue G-250 Stain fluorescence PVDF membranes. Activation of the and destaining Dodeca Silver Stain Kit Coomassie Blue G-250, but with a simpler and protein classes such as glycoproteins (Hart et al. 2003) trihalo compounds in the gels adds 58 Da moieties Fluorescent Protein Stains quicker staining protocol and phosphoproteins (Steinberg et al. 2003, Agrawal Silver Stain Plus Kit to available tryptophan residues and is required for and Thelen 2009), which are of special interest to Flamingo Fluorescent ■■ Fluorescent stains — fulfill almost all of the protein visualization. Proteins that do not contain researchers working in the life sciences (examples Zinc Stain Gel Stain requirements for an ideal protein stain by offering tryptophan residues are not detected. include Pro-Q Diamond and Pro-Q Emerald). high sensitivity, a wide linear dynamic range over Copper Stain Oriole Fluorescent Gel Stain The sensitivity of the Stain-Free system is four orders of magnitude, a simple and robust Imaging Systems Silver Stains protocol, and compatibility with mass spectrometry. comparable to staining with Coomassie (Brilliant) Blue for proteins with a tryptophan content >1.5%; ChemiDoc MP System Negative Stains sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%. Gel Doc EZ System Image Lab Software Gel (top) and blot imaged using stain-free technology.

40 41 Electrophoresis Guide Chapter 6: Protein Detection and Analysis Theory and Product Selection

™ Dodeca High-Throughput Stainers ■■ Table 6.2. Bio-Rad imaging system selection guide. Related Literature CCD (charge-coupled device) cameras — operate Dodeca stainers are high-throughput gel staining with either trans-illumination provided by light boxes devices available in two sizes (Figure 6.1): the small size (visible or UV) positioned underneath the gel or blot Bio-Rad Imaging Systems accommodates up to 24 Criterion gels while the large for imaging a variety of stains (Coomassie, silver, Family Brochure, bulletin 5888 size can accommodate up to 12 large-format gels. The fluorescence) or epi-illumination detected using Imaging Fluorescently stainers ensure high-quality, consistent results and colorimetric or fluorescent techniques. Supercooled Stained Gels with Image Lab eliminate gel breakage from excess handling. CCD cameras reduce image noise, allowing Personal Software Quick Start Guide, detection of faint luminescent signals. Bio-Rad’s ChemiDoc™ Gel Doc GS-800 Gel Doc ChemiDoc PharosFX Molecular bulletin 5989 The stainers feature a shaking rack that holds staining Application MP EZ Densitometer XR+ XRS+ PharosFX Plus Imager™ Gel Doc™ EZ system provides four application- trays at an angle to allow air bubbles to escape and Nucleic Acid Detection ensure uniform gel staining by protecting gels from specific trays: a UV tray (for ethidium bromide Ethidium bromide stain 5 4 — 4 4 5 5 — staining of DNA gels and fluorescence imaging), a SYBR® Green I stain 5 4 — 4 4 5 5 — breaking. They are compatible with the following stains: SYBR® Safe stain 5 4 — 4 4 5 5 — white tray (for Coomassie, copper, silver, and zinc Fast Blast™ DNA stain 4 4 5 4 4 — — — ■■ Bio-Safe Coomassie (Brilliant) Blue G-250 stain stains), a blue tray (for nondestructive nucleic acid Protein Detection, 1-D Gels ■■ Coomassie (Brilliant) Blue R-250 stain imaging), and a stain-free tray for direct visualization, Stain-free gels 5 5 — — — — — — Coomassie Blue stain 4 4 5 4 4 2 2 — ■■ Flamingo Fluorescent gel stain analysis, and documentation of protein samples in Silver stain 4 4 5 4 4 2 2 — polyacrylamide gels without staining, destaining, or SYPRO Ruby protein gel stain 4 4 — 4 4 5 5 — Links ■■ SYPRO Ruby protein gel stain gel drying (see Stain-Free Technology box) Flamingo fluorescent gel stain 4 3 — 4 4 5 5 — ■■ Oriole fluorescent gel stain Oriole fluorescent gel stain 5 4 — 5 5 — — — ■■ Laser-based scanners — offer the highest sensitivity, Protein Detection, 2-D Gels High-Throughput Dodeca ■■ Dodeca silver stain kits resolution, and linear dynamic range. They are Coomassie blue stain 4 3 5 3 3 2 2 — Gel Stainers Silver stain 4 3 5 3 3 2 2 — Imaging powerful image acquisition tools for electrophoresis SYPRO Ruby protein gel stain 4 3 — 3 3 5 5 — Criterion Precast Gels gels and blots stained with fluorescent dyes like Flamingo fluorescent gel stain 4 2 — 3 3 5 5 — Though total protein stains yield visible band Flamingo fluorescent gel stain. Lasers can be Oriole fluorescent gel stain 5 3 — 3 3 — — —

TABLE CONTENTS OF Coomassie Stains patterns, in modern laboratory environments, Pro-Q stain 4 2 — 2 3 5 5 — matched to the excitation wavelengths of a multitude electrophoresis patterns (called electropherograms) Cy2, Cy3, Cy5 label 4 — — — — 5 5 — Bio-Safe Coomassie Stain of fluorophores. Bio-Rad’s PharosFX™ systems use Blot Detection are digitized by dedicated image acquisition devices multiple lasers, which enhance application flexibility Stain-free blots 5 5 — — — — — — Coomassie Brilliant and data are analyzed with sophisticated software. Coomassie Blue stain 4 — 5 4 4 2 2 — and allow optimal excitation of single- or multicolor Blue G-250 Stain Once the gels are digitized, the raw data can be Silver stain 5 — 5 4 4 2 2 — fluorescent samples to enable detection of almost SYPRO Ruby protein blot stain* 5 — — — — 5 5 — stored for further reference. Immun-Star™ chemiluminescence 5 — — — 4 — — — Coomassie Brilliant any fluorescent dye or label Chemifluorescence* 5 — — 1 1 4 4 — Blue R-250 Stain Imaging Systems Quantum dot* 5 2 — 2 2 5 5 — Selecting image acquisition devices for the digitization Multiplex fluorescence 5 — — — — 5 5 — Fluorescent Protein Stains of electrophoresis gels depends on the staining Micro- and Macroarray** Detection Radiolabel — — — — — — 5 5 Flamingo Fluorescent technique used (see also Table 6.2): Fluorescence 5 — — 2 2 5 5 — Gel Stain ■■ Densitometers — based on high-performance Immun-Star chemiluminescence 5 — — — 4 — — — Colony Counting Oriole Fluorescent Gel Stain document scanners with minor modifications Colorimetric detection 5 — — 4 4 3 3 — (water-proof, built-in calibration tool), they utilize Fluorescence detection 5 — — 4 4 5 5 — SYPRO Ruby Protein visible light for analysis of electrophoresis gels Isotopic Detection Gel Stain (transmission mode) and blots (reflective mode) Radiolabel — — — — — — 5 5 X-ray film 4 4 5 4 4 3 3 — Silver Stains stained with visible dyes. Bio-Rad’s GS-800™ — Not recommended; 1–5, recommendation level (5 = highest). calibrated densitometer has a linear response up Dodeca Silver Stain Kit * Optimal with low fluorescence PVDF membrane. to 3.0 optical units in gray levels ** With spot diameters ≥400 µm. Links Imaging Systems

GS-800 Calibrated ChemiDoc MP System Densitometer ChemiDoc XRS+ System Gel Doc EZ System Personal Molecular PharosFX and PharosFX Imager System Plus Systems

Gel Doc XR+ System

Fig. 6.1. High-throughput Dodeca gel stainers.

42 43 Electrophoresis Guide Chapter 6: Protein Detection and Analysis Theory and Product Selection

Imaging Software Molecular Weight (Size) Estimation Related Literature log For protein quantitation using PAGE to be of value: A robust software package is required for image SDS-PAGE is a reliable method for estimating the MW ■■ Employ sample preparation procedures that acquisition to analyze data and draw conclusions from molecular weight (MW) of an unknown protein, since avoid nonspecific protein loss due to insolubility, Molecular Weight PAGE applications. Sophisticated gel analysis software the migration rate of a protein coated with SDS is Determination by SDS-PAGE, precipitation, and absorption to surfaces provides a variety of tools that enhance the user’s inversely proportional to the logarithm of its MW. Linear range bulletin 3133 ■■ Ensure all proteins enter the electrophoretic ability to evaluate the acquired data. The software The key to accurate MW determination is selecting separation medium Using Precision Plus Protein adjusts contrast and brightness, magnifies, rotates, separation conditions that produce a linear relationship Standards to Determine resizes, and annotates gel images, which can then be between log MW and migration within the likely MW ■■ Optimize the quality of the electrophoretic Molecular Weight, separation. For example, wavy, distorted bulletin 3144 printed using standard and thermal printers. All data in range of the unknown protein. A protocol for MW the images can be quickly and accurately quantified. estimation is provided in Part II of this guide. protein bands and comigration of bands lead Molecular Weight Estimation The software can measure total and average quantities to questionable results Using Precision Plus Protein To ensure accurate MW determination: R ■■ WesternC Standards on and determine relative and actual amounts of protein. f When possible, separate a dilution series of pure ■■ Criterion Tris-HCI and Criterion Gel imaging software is also capable of determining the Separate the protein sample on the same gel with a Fig. 6.3. Typical characteristics of a log MW vs. R proteins in parallel. This enables the creation set of MW standards (see Chapter 3 for information f XT Bis-Tris Gels, bulletin 5763 presence/absence and up/down regulation of proteins, curve for protein standards. of a calibration curve (as for molecular weight regarding selection of protein standards) Molecular Weight Estimation their molecular weight, pI, and other values. For more determination with SDS-PAGE, above) and Quantitation of Protein information on imagers and gel evaluation software, ■■ For statistical significance, generate multiple data Quantitation ■■ Analyze all samples (including samples for Samples Using Precision Plus Of all the methods available for protein quantitation visit www.bio-rad.com. points (>3 lanes per sample) calibration) at least in duplicate Protein WesternC Standards, (including UV at 280 nm, colorimetric ■■ Use a sample buffer containing reducing agents the Immun-Star WesternC Bio-Rad offers three different software packages for dye-based assays, and electrophoresis in combination ■■ Use a stain that offers sufficient sensitivity and (DTT or bME) to break disulfide bonds and minimize Chemiluminescent Detection gel imaging and analysis: with image acquisition analysis), only protein quantitation a high dynamic range. Fluorescent stains like Kit, and the Molecular Imager the effect of secondary structure on migration ■■ ™ Flamingo and Oriole fluorescent gel stains are ChemiDoc XRS Imaging Image Lab software — image acquisition and by electrophoresis enables evaluation of purity, yield, ■■ Include SDS in the sample buffer. SDS denatures System, bulletin 5576 analysis software that runs the ChemiDoc MP, or percent recovery of individual proteins in complex recommended over Coomassie and silver secondary, tertiary, and quaternary structures by staining techniques

TABLE CONTENTS OF Gel Doc XR+, Gel Doc EZ, and ChemiDoc XRS+ sample mixtures. imaging systems. The software allows automatic binding to hydrophobic protein regions. SDS also Two types of quantitation are possible: relative quantitation configuration of these imaging systems with confers a net negative charge on the proteins, which (quantitation of one protein species relative to the quantity appropriate filters and illumination sources. It also also results in a constant charge-to-mass ratio of another) and absolute quantitation (quantitation of a allows manual or automated analysis of PAGE gels After separation, determine the relative migration protein by using a calibration curve generated by a range and western blots distance (Rf) of the protein standards and of the of known concentrations of that protein). Because proteins ■■ ® Quantity One 1-D analysis software — acquires, unknown protein. Rf is defined as the mobility of a interact differently with protein stains, the staining quantitates, and analyzes a variety of data, including protein divided by the mobility of the ion front. Because intensity of different proteins at identical protein loads radioactive, chemiluminescent, fluorescent, and the ion front can be difficult to locate, mobilities are can be very different. Thus, only relative quantitative color-stained samples acquired from densitometers, normalized to the tracking dye that migrates only values can be determined in most cases. Absolute protein storage phosphor imagers, fluorescence imagers, slightly behind the ion front: measurements can only be made if the protein under and gel documentation systems. The software investigation is available in pure form and is used as allows automatic configuration of these imaging Rf = (distance to band)/(distance to dye front) the calibrant. systems with appropriate filters, lasers, LEDs, and other illumination sources. It also allows manual or Using the values obtained for the protein standards, plot

automated analysis of PAGE gels and western blots a graph of log MW vs. Rf (Figure 6.3). The plot should

Links ■■ PDQuest™ 2-D analysis software — used for 2-D gel be linear for most proteins, provided that they are fully electrophoretic analysis denatured and that the gel percentage is appropriate for the MW range of the sample. The standard curve is Image Lab Software Analysis sigmoid at extreme MW values because at high MW, Imaging Systems Beyond protein band patterns, PAGE can yield the sieving affect of the matrix is so large that molecules information about a protein’s size (molecular weight) are unable to penetrate the gel. At low MW, the sieving Gel Doc XR+ System and yield (quantity). Image analysis software greatly effect is negligible and proteins migrate almost freely. Links Gel Doc EZ System enhances and facilitates these measurements. To determine the MW of the unknown protein band, interpolate the value from this graph. Fluorescent Protein Stains ChemiDoc MP System The accuracy of MW estimation by SDS-PAGE is in the Flamingo Fluorescent Gel Stain ChemiDoc XRS+ System range of 5–10%. Glyco- and lipoproteins are usually not Oriole Fluorescent Gel Stain Quantity One 1-D fully coated with SDS and will not behave as expected Analysis Software in SDS-PAGE, leading to false estimations. For more Coomassie Stains details about molecular weight estimation using PDQuest 2-D Silver Stains Analysis Software SDS-PAGE, refer to bulletin 3133.

44 45 47 Theory and Product Selection Theory Product and Chapter 7: Downstream Applications 7: Chapter

CHAPTER 7 Downstream Applications the electrophoresis, Following (proteins blotted be might gel entire dried, or a membrane) to transferred excised be might proteins individual or analysis. for gel the from eluted or

46 Electrophoresis Guide

TABLE OF CONTENTS 49 Links Whole Gel Eluter and Mini Whole Gel Eluter EXQuest Spot Cutter PDQuest 2-D Analysis Software Quantity One Analysis 1-D Software Stains Silver Stains Coomassie Stains Protein Fluorescent Fluorescent Flamingo Stain Gel Fluorescent Oriole Stain Gel SYPRO Ruby Protein Stain Gel Theory and Product Selection Theory Product and , and ™ 2-D analysis analysis 2-D , Oriole , spot cutter ™ ™ ™ 1-D analysis 1-D software, and ® Chapter 7: Downstream Applications 7: Chapter SYPRO RubySYPRO protein stain) Freestanding, plastic- or glass-backed 2-D and 1-D 1-D and 2-D glass-backed or plastic- Freestanding, gels SDS-PAGE blots membrane nitrocellulose and PVDF Gels or membranes stained for proteins with visible stains (such as silver and Coomassie blue stains) or Flamingo as (such stains fluorescent ■ ■ ■ Spot Excision (Cutting) software or Quantity One ■ ■ ■ Fig. EXQuest 7.5. spot cutter. Bands containing proteins interest of can beexcised from gels either hand by (for example, using razor a blade) or with the help automatedof spotcutting systems, such as those commonly used in 2-D in proteins The workflows. proteomics gel-based the bands can then beeither eluted from the gel piece (for example, electroelution) by or subjected to downstream processing(for example, tryptic digestion) while still in the gel. Automated spot cutting instruments (spot cutters) are used excise protein to spots interest of from gels and transfer to them microplates to or other vessels EXQuest further Bio-Rad’s for analysis. (Figure 7.5) locates and excises(Figure protein bands 7.5) or spots gels and 2-D from or blots 1-D and loads them into 96- and 384-well microplates or 96-tube racks for downstream processing and analysis. The spot cutter can be fully integrated with the PDQuest it works withthe imaging visualize system to gels and blots that are either visibly or fluorescently stained. In applications, PAGE 2-D PDQuest software tracks the protein bands or spots through spot cutting and mass spectrometric protein analysis. The EXQuest spot cutter allows the use the of following methods: staining and separation common Elution core chamber Cellophane papers Filter Base with bottom electrode µg Mini Whole Gel Eluter 6.5 x 5.5 cm x 0.5 ml 14

mg Whole Gel Eluter 14 x 16 cm x 16 14 30 x 3 ml

Fig. Whole 7.4. gel eluter and mini whole gel eluter elute protein electrophoretically are Proteins fractions. liquid discrete into bands eluted from the gel into precisely spaced channels of the elution chamber core. Liquid fractions are then harvested into a harvesting box. Safety lid with electrode top papers Filter Preparation gel The whole gel eluter and mini whole gel eluter (Figure protein collect and elute simultaneously 7.1) Table 7.3, fractions from preparative slab gels liquid into fractions crude of screening include Applications 7.4). (Figure mixtures proteins of identifyto immunologically relevant and purificationof multiple bandsof nucleic acids or proteins. Fig. Whole 7.3. gel eluter and mini whole gel eluter. specifications. eluter gel Whole 7.1. Table Gel area Fraction volume load Sample

gel ™ cell older (or ® II or Mini-PROTEAN 3 cells) elute to ® Fig. 7.2. Model 422 electro-eluter 422 Model 7.2. Fig. Gel Drying Gel Before the advent modern of gel imaging equipment, gels were dried in a format that not only preserved them, but made them more amenable densitometry to GelAir and 583 Model The autoradiography. or Electroelution Electroelution, as its name implies, is a technique that enable to electrophoresis of principles the applies recovery (elution) molecules of such as proteins from gels and gel slices. It can be used with either slices from a gel containing the protein interest of or with entire preparative gels. Electroelution uses an electrical field and the charged natureof to proteinsmove them from the gel andbuffer a into solution. Once eluted, proteins can be assayed for activity, applied to subsequent purification steps, or subjectedto mass spectrometry or a varietyother of applications. The Model electro-eluter 422 combines with (Figure 7.2) the tank and lid the of Mini Trans-Blot Mini-PROTEAN macromolecules from single or multiple gel slices. The The slices. gel multiple or single from macromolecules electro-eluter has six vertical glass tubes connecting the upper and lower buffer chambers. A frit at the bottom each of tube retains the gel slice but permits current when through migrate to macromolecules passed have macromolecules the When applied. is cap membrane the in collected are they frit, the through for further analysis or testing. Depending on the buffer system, the Model electro-eluter 422 can be used for elution or dialysis six up of to samples. Bio-Rad offers a complete range products of for blotting, including blotting cells for protein transfers, blotting membranes, filterpremixed paper, blotting buffers,reagents, protein standards, and detection kits. Please refer the Protein to Blotting Guide (Bio-Rad bulletin for more information. 5294) dryer systems dry gels between two sheets of cellophane. Wash Wash

based on based conjugated conjugated Transfer Incubate with Incubate Block unbound Block membrane sites membrane primary antibody Develop signal Develop Incubate with Incubate color chemiluminescencecolor secondary or antibody Proteins are transferred from the gel a membrane to where they become immobilized as a replica the of band patterngel’s (blotting). membrane the on sites protein-binding Unoccupied are saturated prevent to nonspecific bindingof (blocking). The blot is probed for the proteins interest of with specific primary antibodies. Secondary antibodies, specificfor the primary antibody type and conjugated detectable to reporter groups such as enzymes or radioactive isotopes, are used label to the primary antibodies. Labeled protein bands are visualized the by bound reporter groups acting on an added substrate or by radioactive decay.

Fig. 7.1. Western blottingFig. 7.1. workflow. Western Blotting (Immunoblotting)Western When specific antibodies available,are transferring the proteins a membrane to (blotting) followed by immunological staining is an attractive complement generalto protein stains and provides additional experiment typical A immunoblotting information. Following PAGE: consists7.1). five of steps (Figure 1. 2. 3. 4. 5.

Mini-PROTEAN Cell Mini-PROTEAN Mini Trans-Blot Cell Model 422 Electro-Eluter 422 Model GelAir Drying System Drying GelAir Model 583 Gel Dryer Gel DryingGel Equipment Links 48 bulletin 2032 bulletin Reagents Brochure, Brochure, Reagents Western Blotting Detection bulletin 2895 bulletin to Transferto and Detection, Protein BlottingGuide, A Guide Related Literature Related Electrophoresis Guide

TABLE OF CONTENTS 51 Methods

Part II Methods

50 Electrophoresis Guide

TABLE OF CONTENTS 53 cells; store 7 Methods

RIPA solubilization buffer solubilization RIPA (use 1 ml buffer RIPA with 3 × 10 SDS-PAGE sample buffer (2x) Centifuge Sonicator Phosphate buffered Phosphate saline (PBS) and use buffer RIPA at 4°C Protein Assay Protein ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Equipment Reagents Links DC Assay RC DC Protein Buffer Sample SDS-PAGE MicroRotofor Cell Lysis Kit (Mammal) ReadyPrep 2-D Cleanup Kit Carefully remove (decant) culture medium from cells. Wash cells twice in cold PBS. bufferAdd RIPA the cells to and keep on ice for 5 min. Swirl the plate occasionally spread to the buffer around the plate. Use a cell scraper collect to the lysate and transfer a to tube. microcentrifuge 1 2 3 Monolayer CulturedCells Monolayer Place the cell suspension on ice, incubate 5 min, and sonicate at Check lysis intervals. appropriate efficacyby light microscopy.  Centrifuge × g for cell debris at ~14,000 and min transfer at 4°C 15 supernatant a new vial.to Perform a protein assay the of supernatant. A protein concentration of 3–5 µg/µl is best for PAGE. sampleAdd 2x SDS-PAGE buffer to the protein solution sample yield to a 1x buffer concentration.

6 7 4 5 cell lysis kit (mammalian) or the protocol detailed here, which uses sonication and ™ Pelletthe cells by at 2,000 × g for 5 min at 4°C. Discard the supernatant and wash pelleted cells in cold PBS. Repeat steps 1 and 2 twice. bufferAdd RIPA the pelleted to cells and suspend the pellet with a pipet. 1 2 3 Protocols Sample Preparation Cells Human MicroRotofor the Use Cells Cultured Suspension radioimmunoprecipitation assay buffer, for cell (RIPA) lysis and protein extraction.

protein assays, ™ ME) or 5–10 mM ME) or 5–10 b or RC DC ™ 2-D cleanup 2-D kit) diminish to ™ carbohydrate content 70°C for 10 min) after for addition 10 70°C sample of buffer for more molecular interactions of disruption complete When preparing sample SDS-PAGE buffer, use either mM) 2-mercaptoethanol ( (~100 5% The final protein concentration in the sample solutionfor electrophoresis1-D should not mg/ml be <0.5 For long-term sample storage, store aliquots at –80°C; avoid repeated thawing and freezing protein of samples Highly viscous samples likely have a very high DNA or carbohydrate content. Fragment DNA with ultrasonic waves during protein solubilization or adding by endonucleases like benzonase. Use protein precipitation with TCA/acetone (for ReadyPrep the with example, When a sample preparation protocol calls for a dilution, the two parts are statedlike but a ratio, what is needed is a fraction. For means take 1 part to one of example, reagent “Dilute and 1:2,” mix with 1 part essentially another, of diluting the part half. by means take 1 part to and mix with“Dilute 3 parts, 1:4,” making 4 parts, of a total diluting the part quarter a by Prepare sample SDS-PAGE buffer without reducing agent, then aliquot and store at room temperature Prepare fresh reducing agent, and add SDS-PAGE it to use before buffersample immediately Dissolve dry protein samples directly sample in 1x buffer; prepare other protein samples such that the final sample buffer concentration is 1x Incubate samples in sample buffer for 5 min at 95°C (or at (DTT) dithiothreitol Solubilize proteins in a buffer that is compatible with the technique electrophoresis corresponding Dissolve pelleted protein samples sample in 1x buffer Dilute dissolved protein samples with sample buffer stock solutions a final to sample buffer concentration1x of Perform a proteinquantitation assay determine to the amount of proteintotal in each sample. Use a protein assay that is tolerant chemicalsto in your samples. For samples in Laemmli sample buffer, for example, use the DC Dilute or concentrate samples as needed yield to a final protein concentration mg/ml >0.5 of Use protein extracts immediately or aliquot them into appropriately sized batches and store them at –80°C avoid to freeze-thaw cycles which detergent. can Omit tolerate 10% up the to protein assay if limited. is amount sample ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Preparation PAGE for Protein Solubilization Protein

) If protein phosphorylation be isstudied, to include vanadate and fluoride as such inhibitors phosphatase Add a chemical protease inhibitor the lysis to buffer. fluoride phenylmethylsulfonyl Examples include fluoride sulfonyl aminoethyl-benzene (PMSF), ketone chloromethyl lysine tosyl (AEBSF), (TPCK), tosylphenylchloromethyletone (TLCK), benzamidine, (EDTA), acid ethylenediaminetetraacetic example, (for inhibitors protease peptide and leupeptin, pepstatin, aprotinin, and bestatin). For best protease a in inhibitors of combination a use results, cocktail inhibitor Perform cell disruption at low temperatures to diminish enzymatic activity samplesLyse using at pH >9 either sodium carbonate as a bufferingor Tris agent in the lysis solution (proteases are often least active at basic pH) Disrupt the sample or place freshly disrupted samples agents denaturing strong containing solutions in such M urea, as 7–9 2 M thiourea, SDS. or In this 2% environment, enzymatic activity is often negligible ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Cell Disruption (

Following cell disruption, check the efficacyof cellwall disruption light by microscopy and centrifuge all extracts remove to any extensively min at 15°C) (20,000 x g for 15 insoluble material; solid particles may block the pores the of gel electrophoresis Optimize the power settings mechanical of rupture systems and incubation times for all lysis approaches. Because mechanical cell lysis usually generates heat, employ cooling where required avoid overheating to the of sample When working with a new sample, use atleast two different cell disruption protocols and compare the protein yield (by protein assay) and qualitative protein content (by SDS-PAGE) To diminish endogenousTo enzymatic activity: Suspend ~1 mg (wet weight) pelleted cells in ~10 µl mg weight) (wet pelletedSuspend cells ~1 in ~10 of concentration for a protein sample buffer SDS-PAGE tissue in liquid nitrogen, 3–5 µg/µl. If disrupted samples like liver biopsies and plant leaves contain and 10–20% protein, respectively1–2%

■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Lysis General Tips Sample for Preparation the (increasing simple workflow preparation sample the Keep number sample of handling steps may increase variability). Protocols Sample Preparation

52 Electrophoresis Guide

TABLE OF CONTENTS

55

Methods Reproducible lysis and and lysis Reproducible protein solubilization of bacteria and yeast is the because challenging cells may release proteases and other enzymes into the growth medium. Wash the with thoroughly cultures buffers take and isotonic precautions inactivate to the proteolytic activity after cell disruption Extensive lysis. of microbial cells is required, usually with the help of a French press, bead impact sonicator or instruments, SDS SDS sample buffersolubilization SDS-PAGE sample (2x) buffer Phosphate buffered saline (PBS) Centifuge Sonicator ■ ■ ■ ■ ■

■ ■ ■ ■ ■ MicroRotofor Cell Lysis Kit (Bacteria) MicroRotofor Cell Lysis Kit (Yeast) SDS-PAGE Sample Buffer (2x) Bio-Spin and Micro Bio-Spin Columns ReadyPrep 2-D Cleanup Kit n Tips Equipment Links Reagents 2-D cleanup cleanup 2-D ™ or Micro ® columns, which are filled with size ™ Buffer exchange — use Bio-Spin use — Buffer exchange Precipitation — use the ReadyPrep exclusion media equilibrated buffer. These in Tris μl 50–100 volume sample a accommodate columns and remove compounds kD <6 min. within 10 Mix the purified sample with 2x SDS-PAGE sample buffer Bio-Spin for precipitation) acetone/TCA an on (based kit interfering substances and of simultaneous removal concentration dilute of samples ng/ml) (<50 ■ ■ ■ ■ from Chromatography Fractions Protein When checking fraction purity or the enrichment a of separation, chromatographic a after particular protein you can observe the presence high of concentrations salt,of detergent, denaturants, and organic solvents. proteins chromatography, exchange ion in example, For are eluted a salt by gradient. But the salt concentration theof corresponding fractions can be ashigh M, as 0.5 a concentration that interferes with SDS-PAGE. Remove salt and other contaminants one by the of approaches: following

) for 3 min at 7 5,000 x g and resuspend the pellet in an equal PBS and volume 37°C of centrifuge again. Repeat two more times remove to all interfering material (extracellular proteases and growth media). Add 200 SDS sample µl hot of (95°C) solubilization buffer the pellet to and thoroughly. vortex Sonicate the sample solution times 10 for 1 sec each kHz. at ~60 W and ~20 Incubate for 5 min. at 95°C  Cool the sample and 20°C to dilute with sample µl 2x SDS-PAGE buffer. ~250 Incubate for another min 20 at room temperature. Centrifuge the sample solution at 20°C x g andfor harvest 30 min at 14,000 the supernatant. Perform the The protein assay. protein concentration should be µg/µl. ~5 Centrifuge x 10 cells (~5 5 6 1 2 3 4 Protocols Sample Preparation Microbial Cultures Use the MicroRotofor cell lysis kit (bacteria) or the MicroRotofor cell lysis kit (yeast). Alternatively, use the protocoldetailed here, which relies on cell lysis with ultrasonic waves in combination with a solubilization in SDS under elevated temperature. This ensures deactivation and denaturation proteases. of

Cool protein precipitation and wash solutions Chill –20°C. to mortar a with nitrogen. liquid Place 3–4 leaves in the mortar, add liquid nitrogen, and grind the leaves in the liquid nitrogen a fine to powder. leafTransfer powder ml 20 protein of into precipitation solution and incubate for Stir1 hr at –20°C. occasionally. Centrifuge the solution at –20°C for min at 35,000 Discard x g. 15 supernatant, add wash solution and suspend the pellet. min Incubate for 15 stirringat –20°C, occasionally. Repeat until wash solution turns light green. Centrifuge the solution at –20°C for min at 35,000 x g and15 discard supernatant. Resuspend pellet in 2 ml wash solution. suspensionTransfer a shallow into ceramic shell and cover with perforated parafilm. Put shell a dessicatorinto and apply a vacuum until the pellet (acetone powder) is dry. Mix 3 mg sample of powder with 1 ml 1x sample buffer incubate and SDS-PAGE for 30 min at room temperature. Vortex from time time. to Centrifuge the solution at –20°C for Collect x g. and heat min at >16,000 15 the supernatant for 3 min at 95°C. Cool solution roomto temperature and perform the protein assay. 1 8 9 4 5 6 7 2 3 Leaves Plant minimize the deleteriousTo effects endogenous of kit lysis cell MicroRotofor the use compounds, plant (plant) or the protocol here, which involves grinding the tissue in a mortar and pestle with liquid nitrogen.

Chill a mortar with liquid nitrogen, then grind small tissue pieces in the presence liquid of nitrogen a to fine powder. Immediately after grinding, transfer 60 mg tissue powder a to containing tube microcentrifuge ml lysis buffer. 1.0 Optional: sonicate the sample on ice 5 times, for 2 sec every time. Pause between sonication steps to avoid overheating. Incubate the sample at room temperature for 30 min. Vortex from time time. to Centrifuge at 35,000 x g for 30 min at room temperature. Perform a protein assay determineto the protein concentration the of supernatant, which µg/µl. should be 5–10 Dilute supernatant with 2x SDS- samplePAGE buffer1x a final (to concentration), and incubate for min20 at room temperature. Do not heat the sample. 7 4 5 6 2 3 1 Mammalian Tissue Use the MicroRotofor cell lysis kit (mammal). Alternatively, use the protocol here, which involves freezing mammalian tissue samples (for example, biopsy samples) in liquid nitrogen at –196°C.

Mortar and pestle Protein precipitation precipitation Protein solution Wash SDS-PAGE sample Lysis buffer SDS-PAGE sample (2x) buffer buffer (1x)

With plant leaves, precipitate precipitate leaves, plant With proteins with in 20% TCA prechilled acetone (–20°C). remove theTo plant phenols, rinse the pellet at least twice with cold acetone (–20°C) and air-dry samples in a vacuum Use liquid nitrogen and a mortar and pestle grind to the samples while they are still frozen. Mill any larger pieces example, (for beforehand wrap the frozen tissue sample in aluminum foil and crush with a hammer) ■ ■ ■ ■ ■ ■

■ ■ ■ ■ ■ ■ Links Reagents

Tips 54 (Plant) MicroRotofor Cell Lysis Kit SDS-PAGE Sample Buffer (Mammal) MicroRotofor Cell Lysis Kit Equipment n n Sample Preparation Protocols Electrophoresis Guide Mammalian Tissue Mammalian solution Plant Leaves Plant

TABLE OF CONTENTS 57 Methods Product Links: Product Use only high-quality reagents, acrylamideespecially monomers, problems polymerization avoid to Proper degassing and filtering of the casting solution is critical for both polymerization the of reproducibility (oxygen removal) and the avoidance of spectrometry mass to related problems contamination) ( A temperature of 23–25°C is best polymerization; and degassing for equilibrate the stock solutions to room temperature should reactions APS/TEMED-initiated proceed for at least 2 hr ensure to size pore of maximum reproducibility Make fresh APS solution every day performance best for Replace TEMED every three months becauseit is subject to the causes which oxidation, gradual loss of catalytic activity The glass plates must be clean and free of chips. Clean glass plates with ethanol and lint-free cloths before use The height of the stacking gel should be at least 2x the height of the sample in the well. This ensures band sharpness, even for diluted samples protein Store gels flat in the fridge 4°C. at Do not freeze. Wrap handcast gels tightly in plastic wrap with combs insertedstill Run handcast gels with discontinuous buffer systems right after gel casting because the buffer discontinuity gradually strength) ionic and (pH disappears during gel storage. SDS- gelsPAGE are not stable at pH 8.8 over a longer time period acrylamide about information more For polymerization, refer Acrylamide to Polymerization – A Practical Approach, bulletin 1156 Acrylamide and bisacrylamide are bisacrylamideAcrylamide and are neurotoxins when in solution. Avoid direct contact with the solutions and clean up spills For casting multiple gels, use the Mini- PROTEAN 3 multi-casting chamber PROTEAN II (catalog #165-4110), (catalog chamber casting multi-gel multi- Plus PROTEAN or #165-2025), #165-4160) (catalog chamber casting

        n n n n n n n n n n n General Tips for Handcasting for Tips General n n — 75 µl 75 7.5 µl 7.5 15 ml 15 150 µl 150 X% 3.75 ml 3.75 0.33 x X ml 20 cm ml 16.0 ml 24.0 ml 32.0 48.0 ml 96.0 ml II xi Cell 11.03–(0.33 x X)11.03–(0.33 ml ®

— 75 µl 75 7.5 µl 7.5 15 ml 15 150 µl 150 6.0 ml 3.75 ml 3.75 16 cm 16 ml 12.8 ml 19.2 ml25.6 38.4 ml 76.8 ml 5.03 ml PROTEAN Resolving Gel Resolving — ™ 75 µl 75 7.5 µl 7.5 12% 7.5% 15 ml 15 150 µl 150 7.28 ml 7.28 3.75 ml 3.75 3.75 ml 3.75

— — ml 15.0 — — Cell Criterion *

® — 9 ml 15 µl 15 4% 75 µl 75 15 ml 15 150 µl 150 3.78 ml 3.78 1.98 ml 1.98 Stacking Gel — 4.2 ml ml5.6 ml8.4 — Cell Mini-PROTEAN O 2 10 ml of monomer10 solution is sufficient for two stacking gels of any thickness. Prepare the resolving and stacking gel solutions without APS or TEMED. (Tables consult 1 and 2; the instruction manual for the system you are using for more details.) Table Volume 1. of resolving gel solution required to fill a gel cassette. Volumes listed are required completely to fill a gel cassette. Amounts may be adjusted depending on the application (with or without comb,with or without stacking gel, etc.). Spacer 0.5 mm mm 0.75 mm 1.0 mm 1.5 mm 3.0 *  (Gel Thickness) Degas the solution under a vacuum min. for While at least solutions 15 sandwich. cassette glass the assemble degassing, are Place a comb the into assembled gel sandwich. place With a marker, a mark on the glass plate 1 cm below the teeth the of comb. This will be the level which to the separating gel is poured. Remove the comb. TEMED volumeTotal diH 10% SDS 10% 1.5M Tris-HCl, pH1.5M 8.8 Table 2. Recipes for stacking and resolving gels. Adjust amounts as needed for the format you are using (see 1). Table 10% APS 10% 0.5M Tris-HCl, pH 6.8 30% Acrylamide/bis 1 3 2 Protocols Gels Polyacrylamide Handcasting Single-Percentage Gels . Each ' to each to tube ' Reagent I into . Reagent each II into ' Reagent each B to Reagent each to S μl 5 Reagent needed. A This solution and vortex. Incubatetubes at room temperature for 5 min, or until the Vortex. dissolved. is precipitate Add 1 ml DC of tube and vortex immediately. Incubate at room temperature min. for 15 Add 5 μl DC of of DC is referred as Reagent to A standard or sample assayed requires μl Reagent A 127 Prepare 3–5 dilutions a protein of mg/ml protein). standard (0.2–1.5 Pipet µl protein of standard 25 or microcentrifuge clean into sample µl RC of tubes. Add 125 each tube and vortex. Incubate the tubes for 1 min at room temperature. µl RC of Add 125 tube and vortex. Centrifuge the tubes x g for 3–5at 15,000 min. Discard the supernatant inverting by the tubes on clean, absorbent tissue Allowpaper. the liquid drain to tubes. the from completely µl Reagent of A Add 127 5 7 1 2 4 6 3 Microfuge Tube Assay Protocol (1.5 ml) Assay Protocol (1.5 Microfuge Tube . ' . ' to each to tube ' Reagent each II to Reagent each to S Reagent each B to tube Read absorbance each of sample nm. The at 750 absorbances are stable for at least 1 hr. Plot absorbance measurements as a function of concentration for the standards. Interpolate the concentration the of protein samples from measurements. absorbance sample and plot the Reagent needed. A This Reagent each I into tube and 8 9 10 ProteinRC DC Assay) and vortex. Incubatetubes at room temperature for 5 min, or until the Vortex. dissolved. is precipitate Add 4 ml DC of and vortex immediately. Incubate at room temperature min. for 15  Add μl DC 20 of 1 ml DC of solution is referred as Reagent to A Prepare 3–5 dilutions a protein of mg/ml protein). standard (0.2–1.5 µlPipet protein of standard 100 or sample clean into tubes. Add 500 µl of RC vortex. Incubate the tubes for 1 min at room temperature. Add 500 µl RC of tube and vortex. Centrifuge the tubes x g for 3–5at 15,000 min. Discard the supernatant inverting by the tubes on clean, absorbent tissue paper. Allow the liquid drain to completely from the tubes. µl Reagent of A Add 510 Each standard or sample assayed μl Reagentrequires A 510 7 5 6 2 3 4 1 Standard Assay Protocol (5 ml)Standard (5 Assay Protocol Plus ™

For best results, prepare the standards in the same sample the buffer as Prepare a standard curve each time the assay is performed

56 SmartSpec RC DC Protein Assay PROTEAN II xi Cell Mini-PROTEAN Cell Mini-PROTEAN Criterion Cell Criterion Links n n standard curve provides a relative measurement protein of concentration. Tips detection reagents a protein solution to and subsequent measurement absorbance of nm with at a spectrophotometer. 750 Comparison a to and detergent compatible Protein quantitation (DC). can be performed in complex mixtures including Laemmli buffer. It involves addition of The RCDC protein assay is based on a modification and is reducingboth of the Lowry agent protocol compatible(Lowry1951), et (RC) al. Sample Quantitation ( Sample Quantitation Protocols Electrophoresis Guide Spectrophotometer

TABLE OF CONTENTS 59 Methods If gravity flow isn’t fast peristaltic a use enough, pump pump to the entire set of gradients within min. If 10 it is not possible complete to the operation min in from 10 the time initiators are added, then it might be necessary reduceto the amount the half (use initiators of amount of TEMED) slow to gradient The polymerization. quickly as poured be should mixing without possible, as the in solution gradient the chamber casting

n Tips Links Mini-PROTEAN 3 Multi-Casting Chamber Model 485 Gradient Former

multi-casting chamber. Place the gradient former on a magnetic stir plate and add a magnetic stir bar to themixing chamber labeled Attach “light.” the luer fittingto the stopcockvalve on the inlet port. Run tubing a piece Tygon of tubing works ID Tygon well) from the (1/8" gradient former the luer to fitting on the multi-casting chamber. Combine all reagents except the initiators, and degas the solution min. for15 Just prior pouring, to add TEMED and bothAPS solutions to and mix gently. solutions monomer appropriate the Pour (Consult chambers. gradient the into manual instruction former gradient the light the Pour instructions.) complete for solution the into mixing chamber labeled and the heavy“light,” solution in the “heavy.” labeled reservoir chamber on the stirringTurn bar in the mixing openchamber, the tubing clamp the of gradient maker and the stopcock valve of the casting chamber, and pour the gels. formulations using the chart on the the Reassemble 4) Table (see right. Determine the chamber volumes. To To volumes. chamber the Determine create a linear gradient, the volume in each chamber is half the gel total volume greater). is whichever ml, 20 (or required As an example, casting mm twelve 1.0 gels requires so ml, prepare ml 100 105 Divide that volume 1). (step two by to determine the volume required for each chamber the of gradient ml former (52.5 each for the light and heavy chambers). Determine the heavy and light acrylamide 5 6 7 2 3 4 Volume Volume 85–90 ml X = 7.3 ml X = 7.3 ml X = 13.8 X = 34 ml µl X = 275 µl X = 27.5 mlX = 36.7 ml X = 13.8 mlX = 4.5 µl X = 275 µl X = 27.5 to Prepare 105–110 ml 105–110 145–150 ml 145–150

80 ml 140 ml 140 100 ml100 for 12 Gelsfor 12 Volume Required Required Volume

Plates mm 0.75 mm 1.0 mm 1.5 Determine the volume acrylamide of prepare mlto is required (≥40 for the Model 485 gradient former). Prepare the required ml) volume of (+5 acrylamide. 3 provides Table estimated volumes for mini- the casting 12 of Mini- of stack the Assemble gels. format PROTEAN 3 cassettes as described in the Mini-PROTEAN 3 multi-casting chamber instruction manual. Then, flowwater through the stopcock and the fill to required volume the measure chamber the Disassemble cassettes. and dry all components. for acrylamide required of Volume 3. Table Prepare gels. mini-format the amount12 listed ml. 5 additional an plus below Spacer Table 4. Preparation of light and heavy acrylamide solutions. Solution (4%) Light Acrylamide stock 30% (30%)(X ml) ml) = (4%)(55 M Tris-HCl1.5 stock buffer, pH 8.8 M)(X ml) M)(55(1.5 = (0.375 ml) Water ml) ml + 13.8 = X (55 ml) – (7.3 10% APS (500 ml) µl)/(100 = (X µl)/(55 ml) TEMED APS volume;10% = X µl)/10 (275 (20%) Solution Heavy Acrylamide stock 30% (30%)(X ml) ml) = (20%)(55 M Tris-HCl1.5 stock buffer, pH 8.8 M)(X ml) M)(55(1.5 = (0.375 ml) Water (55 ml) ml) ml – (36.7 + 13.8 = X APS (500 ml) µl)/(100 = (X µl)/(55 ml) TEMED APS volume;10% = X µl)/10 (275 1 Protocols Gels Polyacrylamide Handcasting Gradient Gels This protocol is for preparing mini-format linear 12 gradient gels. It requires the Model 485 gradient former and Mini-PROTEAN 3 multi-casting chamber. For other protocols, refer the instruction to manual for the gradient former you are using. O.

2

Dry the area above the separating gel with filter paper before pouring stackingthe gel. Place the comb in the cassette and tilt it so that the teeth are at a slight angle. This prevents air from (~10°) becoming trapped under the comb while the acrylamide solution is poured. being Add the APS and TEMED the to solution, gel degassed resolving and pour the solution down the side upturned the nearest spacer theof comb. Pour until all the teeth are covered the by solution. Realign the comb in the sandwich and add monomer fill to the overlay An completely. cassette solution is not necessary for comb a when polymerization is in place. Allow the gel polymerize to 30–45 min. Remove the comb pulling by it straight up slowly and Rinse gently. the wells completely with diH 4 5 6 1 2 3 Pour the Stacking Gel

-butanol. Stacking gel Stacking gelResolving (contd.) O. 2 Add the APS and TEMED the to degassed resolving gel solution, and pour the solution the mark, to using a bulb. and pipet glass Using a Pasteur pipet and bulb, monomer the overlay immediately solution with n water-saturated Allow the gel polymerize to 45–60 min. The gel is polymerized once you see a line form between the stacking and the resolving gel. Pour off the overlay solution and rinse the the of top gel with diH 3 1 2 -butanol). Recalculate your gel casting recipes recipes gel casting your Recalculate -butanol). so that the separation gel solution contains 25% glycerol.(w/v) Due the significant to difference in density, the two solutions mix when won’t the stacking gel solution is carefully poured on of top the resolving gel solution. Alternative Casting Procedure Casting Alternative It is possible cast to separation and stacking gels one after with no another, intermediate step requiring overlay solution (water-saturated n Pour the Resolving Gel Single-Percentage Gels -butanol, or t-amyl -butanol or t-amyl

Do not allow alcohols to remain on the gels for more than 1 hr or dehydration of the top of the gel will occur. convenient sometimes is It castto the separating discontinuous the portion of gel the afternoon before stacking gel the casting and running the gel. If the stacking gel is be to cast the following place day, approximately 5 ml of 1:4 diluted running gel buffer on top of each separating gel after rinsing with deionized water prevent to dehydration of the separating gel For the overlay solution, water, n can also be used. With n solution overlay the alcohol, can be applied rapidly because very little mixing will occur. If using water overlay,to use a needle and syringe and a steady, even rate of delivery to prevent mixing When pouring the resolving resolving the pouring When gel solution, pour the solution down the middle of the outside plate of the gel sandwich or downthe side of one of the spacers. Pour smoothly prevent to it from mixing with air

58 Reagents Polyacrylamide Gel Polyacrylamide Gel Links Tips Handcasting Polyacrylamide Gels Polyacrylamide Handcasting Protocols Electrophoresis Guide n n n

TABLE OF CONTENTS 61 Methods Links Prot/Elec Tips

tipsfit ™ Coomassie Coomassie ™ The total protein amount loaded per lane depends on the sample complexity and sensitivity of the staining technique. Using 15–20 µg protein per lane for mini- or midi-format gels is a good starting point for complex protein samples when staining with Bio-Safe >12,000 x g at 20°C before>12,000 loading remove to insoluble material that may clog the pores of the acrylamide gel For best resolution, load a concentrated sample rather than a diluted amount Centrifuge the sample solution min at for 10–15 avoid edgeTo effects, sample add 1x buffer unused to wells and never overfill wells Load samples either before or after placing the methods Both tank. the into modules electrophoresis produce acceptable results. In both cases, fill both the assembly (inner chamber) and the tank (outer chamber) with buffer Add running buffer the to cathode buffer reservoir first and then apply the sample on the stacking gel under the electrode buffer. Sample buffer must contain glycerol to stabilize the sample application zone in the sample well of the gel Use pipet tips designed for protein sample loading for bestresults. For example, Bio-Rad’s Prot/Elec If using Bio-Rad’s patented sample loading guide, place it between the two gels in the electrode assembly. Sample loading guides are available and 15-well 12, for 10, 9, formats. Use the sample loading guide locate to the sample wells. Insert the Hamilton syringe or pipet tip into the slots of the guide and fill the corresponding wells Load samples slowly allow to them settle to evenly on the bottom of the well. Be careful not puncture to the bottom of the well with the syringe needle or pipet tip easily between vertical slab gel plates mm while of 0.75 maintaining a large bore for fast flow of sample stain. Determine the optimum protein load by running a sample the of series dilution ■ ■ ■ ■ ■ ■ ■ ■ ■ Tips for Sample Loading Sample for Tips ■ ■ ■ ■ ■ ■ ■ ■ ■

band smiling, and lane distortions lane and smiling, band When preparing running buffers, make the solution as specified in the protocol and do not titrateto a pH. The ion balance is set by the concentration of reagents; adjusting the pH alters this balance and leads undesirable to results buffers running reuse not Do per V Use 5–10 cm of gel for about min during 10 sample entry (or until the sample has concentrated at thestarting point of the separation gel). Then continue with the voltage setting recommended inthe instruction manual for the using are you system electrophoresis Use the voltage setting recommended in the instruction manual for the electrophoresis system you are using; resolution, band decreased to leads voltage excessive When running multiple cells, use the same voltage for multiple cells as you would for one cell. Be aware that with double will supply power the by drawn current the temperature the maintain reproducibility, maximize To of the electrophoresis buffer with at the 15°C help of a cooler recirculating two – compared one to – cells. Use a power supplythat can accommodate this additive current and set the current function additive this permit to enough high limit ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ Tips for Electrophoresis for Tips Protocols Performing Electrophoresis

— 5 µl 5 µl 10 µl 10 Nonreducing Initial 35–50 mA Final 20–31 mA 5 µl 10 µl 10

4.75 µl 4.75 0.25 µl Reducing

O. 2

Sample buffer: Use Laemmli sample buffer. Remove the comb and tape from the gels and assemble the cell. electrophoresis Fill the inner and outer buffer chambers with running buffer. Fill the upper (inner) buffer chamber each of core with 200 ml of running1x buffer. Fill the lower (outer) buffer chamber the to indicator mark for 2 gels ml) (550 or 4 gels (800 of ml) with 1x running fill the outer runsbuffer buffer. At V, >200 chamberto the 4-gel (800 ml) mark. Running buffer (1x): Dilute 100 ml of 10x stockRunning Dilute ml 10x of 100 buffer (1x): with 900 ml diH -mercaptoethanol gel in the Mini-PROTEAN cell. For detailed Tetra instructions, Total volumeTotal Laemmli sample buffer b b. Prepare gels and assemble the electrophoresis cell: a. b. Prepare samples as indicated in the table below. Component Sample min). 10 for 70°C Heatsamples at (or min for 90–100°C 5 at Load the appropriate volume your of protein sample on the gel. Connect the electrophoresis cell the power to supply and perform electrophoresis according the following to conditions: Run conditions: 200 V minRun 31–39 time: Expected current (per gel): These conditions gels. are SDS-PAGE If using for Tris-HCl Prepare buffers: a. Bio-Rad’s Mini-PROTEAN gels, TGX the gels can be run at 300 decrease V to the run time. After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel floating by offit the water. plate into Stain and image the gel, using one the of protocolson the following pages as examples. as pages following ™

TGX ®

7 3 4 5 1 2 6

-Mercaptoethanol 60 PowerPac Basic Power Supply Laemmli Sample Buffer Sample Laemmli b Running Buffer Buffer Running Mini-PROTEAN Tetra Cell Tetra Mini-PROTEAN Mini-PROTEAN TGX Gels Precast Links

The following is a generalized protocol for running a Mini-PROTEAN General Protocol: SDS-PAGE SDS-PAGE General Protocol: Performing Electrophoresis Protocols Electrophoresis Guide refer the Mini-PROTEAN to precast gels instruction manual and application guide (bulletin #1658100).

TABLE OF CONTENTS 6363 Methods Migration distance of of distance Migration dye front (67 mm) Links Quantity One Analysis 1-D Software Precision Plus Protein Standards Unstained Criterion Tris-HCl Precast Gels Stain Coomassie Bio-Safe 4–20% Tris-HCI gel and Product Links: Product was generated using the ™ f > 0.99) between the proteins’ MW 2 Unknown band Unknown Top of resolvingTop gel Migration distance of of distance Migration unknown band (45 mm) Precision Plus Protein standards from Figure The strong 1. linear relationship (r Fig. 2. Determining the MW of an an of MW the Determining 2. Fig. unknown protein by SDS-PAGE. A standard curve of the log (MW) versus R and migration distances demonstrates distances demonstrates migration and MW. predicting in reliability exceptional 1.0 2 min 10 min 10 10 min 10 10 min 10 10 min 10 30 min 60 min ~5 min ~5 min ~5 min 130 min 130 min 130 >1.0 mm Gel >1.0 Standards Unknown 0.8 0.6 f R 5 min 5 min 5 min 1 min 5 min 15 min 15 min 15 20 min 30 min ~5 min ~5 min ~5 min 0.4 0.5–1.0 mm Gel brown precipitate appears. precipitate brown 0.2 = 0.997 1 2 3 4 5 6 7 8 2 Duration y = –1.9944x + 2.7824 y = –1.9944x r

unstained standards; lanes 2–8, a dilution series of an E. coli lysate containing a hypothetical — — ™ 3 min 2 min 2 min 2 min 15 min 15 min 15 15 min 15 30 min ~5 min ~5 min 0 15 10 75 37 25 20 50 0

150 100 250

1.0 2.0 3.0 <0.5 mm Gel log MW log MW, kD Fig. Example 1. showing MW determination of an unknown protein. μl of Lane Precision 10 1, Plus Protein stained with Bio-Safe Coomassie stain. Gel shown is the actual size. unknown protein (GFP). Proteins were separated by SDS-PAGE in a Criterion

~30 sec. Develop until solution turns yellow or until

graphically f 200 ml 200 ml 200 ml 200 ml 200 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml Volume

. f value using the the using value f O O analysis software (or equivalent). (or software analysis 2 2

®

Stop diH diH Fixative Fixative Oxidizer Reagent Developer = migrationdistance the of protein/ f Silver reagent 5% acetic5% acid following equation: following R migration distance the of dye front Repeat this step for the unknown bands in the samples. log the plot program, graphing a Using (MW) as a function R of Generate the equation and y = mx + b, solve determine for y to the MW the of protein. unknown Using a ruler, measure the migration migration the measure ruler, a Using distance from the the of top resolving gel each to standard band and the to front. dye For each band in the standards, R the calculate 10% ethanol/5%10% acetic acid 40% methanol/10% acetic acid yellow color is gone from the gel) (Repeat washes 5–7 times until all the 1 2 3 4 5 3 4 2 1 8 6 7 11 12 13 10

5

9

StainingSilver (Bio-Rad Stain) Silver Protocols Estimation Molecular Weight Run the standards and samples gel. on an SDS-PAGE Process the gel with the desired stain and then destain to visualize the protein bands. Determine the R Figures 1 and 2 illustrate the procedure. graphically: MW determine To Step

or using Quantity One

O).

2 O prior to imaging. to prior O 2 and agitate gently min. for 10 Rinse gelwith diH Cover the tray, placeCover on the a rocker tray, or shaker and agitate Stain gently. for at least 3 hr. (Optional) Carefully pour off the stain solution and replace with an equal Cover 20. Tween (w/v) volume 0.1% of place onthe a rocker tray, or shaker Place gel in a staining ml tray with of 100 fixing aceticsolution10% ethanol,(40% place acid). Cover on the a rocker, tray, and agitate gently for at least 2 hr. Pour off the fix solution and add 50 ofml stain solution1x (dilute 1 part Flamingo fluorescent gel stain with 9 parts diH Fluorescent Gel Stain ™ 2 3 4 1 Flamingo

O prior to to prior O 2 O for min. ≥30 2 O per gel. 2 Remove all water from staining container and add 50 ml Bio-Safe of Coomassie stain enough (or completely to cover gel). Agitate for 1 hr. Rinse in 200 ml diH If using the 5 L configuration, prepare Oriole stain solution adding by mix well shaking. by Place gel in a staining tray with 50 ml of Oriole fluorescent gel stain. Cover the place and agitate on a rocker, tray, hr. gently for ~1.5 the gelTransfer diH to Wash gels three times for 5 min each 400 ml methanol of the 1 L bottle to diluents.of Then ml Oriole of add 10 fluorescent gel stain concentrate and in 200 ml diH imaging. Destaining is not necessary. not Destaining is imaging. Stained gelscan be stored in water. Coomassie Stain ™ Fluorescent Gel Stain ™ 3 2 3 1 1 2 Total Protein Staining Protein Total General protocolsare described below gels. For Mini-PROTEAN for more details, refer the instruction to manual for the stain you are using. Bio-Safe Protocols Oriole O 2 drying system ™

For long term-storage, shrink-wrap the stained gels glycerolin a 10% solution (storage at or 4°C) dry them with a GelAir Fluorescent dyes like Oriole and Flamingo fluorescent gel stains have a higher dynamic range than Coomassie or silver staining techniques, making them quantitative for suitable analysis protein Use pure chemicals and diH purified highly Gently agitate the container on a horizontal shaker, making sure the gel is completely covered with stain solution all the time Use clean and dust-free containers for gel staining. If possible, place a lid on the container avoid to contamination of the solution staining Always weargloves during the staining process. to Try avoid touching the gels with gloves Wet fingers. your with water or buffer before handling the gel keep to the gel from sticking and tearing Gels stained with fluorescent counterstained be can dyes Coomassie colloidal with furtherfor reference. In enhances so doing fact, colloidal the of sensitivity stain Coomassie (conductivity <2 μS) <2 (conductivity

within 60 min

6262 GelAir Drying System Drying GelAir Silver Stains Silver Oriole Fluorescent Fluorescent Oriole Stain Gel Flamingo Fluorescent Fluorescent Flamingo Stain Gel Fluorescent Protein Stains Protein Fluorescent Bio-Safe Coomassie Stain Coomassie Bio-Safe Coomassie Stains Coomassie Mini-PROTEAN Gels Links n n n n n n n 

Electrophoresis Guide Electrophoresis Guide       TIPS

TABLE OF CONTENTS 65 Methods

6.0 ml 6.0 6.0 ml 6.0 0.3 ml ml 0.3 0.3 ml ml 0.3 12.0 ml 12.0 3.75 ml 3.75 3.75 ml 3.75 15.0 ml 15.0 24.0 ml 24.0 ml 24.0 to 30 mlto 30 mlto 30 mlto 12.00 mg 12.00 -mercaptoethanol -mercaptoethanol

-mercaptoethanol 950 μl (50 μl to sample buffer) -mercaptoethanol 980 μl to μl (20 sample buffer) -mercaptoethanol (added fresh) O O O 2 2 2 Sample Buffers 2x SDS-PAGE (Laemmli,30 ml) (catalog #161-0737) glycerol, SDS, 25% pH 2% 6.8, mM Tris-HCl, 62.5 b 5% blue, bromophenol 0.01% fresh) (added pH 6.8 M Tris-HCl, 0.5 Glycerol50% 1.0% Bromophenol blue blue Bromophenol 1.0% SDS 10% diH Add b use. before 2x Native (30 PAGE ml) (catalog #161-0738) glycerol, pH 40% 6.8, mM Tris-HCl, 62.5 blue bromophenol 0.01% pH 6.8 M Tris-HCl, 0.5 Glycerol50% blue Bromophenol 1.0% 2x Tricine (30 ml) (catalog #161-0739) SDS, glycerol, 40% pH 2% 200 6.8, mM Tris-HCl, Coomassie0.04% Brilliant Blue G-250, 2% b pH 6.8 M Tris-HCl, 0.5 Glycerol50% SDS 10% Coomassie Blue G-250 diH Add b before use. diH

1 ml 5 ml 50 ml 80 ml 60 ml 90 ml 0.10 g 0.10 2.40 g 2.40 6.00 g 27.23 g 27.23 87.60 g 87.60 10.00 g 10.00 to 150 ml 150 to to 100 ml 100 to to 100 ml 100 to to 300to ml

-Butanol , 1 L) , 1 L) O O O O O O O O O 2 2 2 2 2 2 2 2 2 -Butanol catalog #161-0799 catalog #161-0798 Store at 4ºC. 0.5 M Tris-HCl, pH 6.8 ( diH N'N'-bis-methylene-acrylamide Buffer Formulations GelCasting Reagents Acrylamide/Bis 2.67% (30% T, C) Acrylamide ml) g/100 (29.2 10% (w/v)10% APS (fresh daily) (catalog #161-0700) persulfate Ammonium diH Filter and store at 4ºCin the dark (30 days). Premade alternatives: powder acrylamide/bis 37.5:1 Catalog #161-0125, 30% acrylamide/bis solutionCatalog #161-0158, 1.5 M Tris-HCl, pH 8.8 ml) (150 ( ml) g/100 base (18.15 Tris diH Adjust pH with 8.8 to 6 N HCl. diH base Tris diH Adjust with pH 6 N HCl. 6.87 to diH Store at 4ºC. (w/v)10% SDS (100 ml) (catalog #161-0416) SDS diH Dissolve with gentle stirring diH n Water-Saturated Combine in a bottle and shake. Use the phase top only. Store at room temperature. n diH

40 ml 80 ml 80 ml 1.0 ml 1.0 1.0 ml 1.0 ml 1.6 2.0 ml 2.0 7.60 g 7.60 2.00 g 2.00 0.50 g 0.50 0.50 g 0.50 0.20 g 0.20 g 0.20 0.60 g 0.60 2.92 ml 2.92 0.08 ml ml 0.08 21.00 g 21.00 20.00 g 20.00 to 50 ml to to 50 ml to to 45 mlto to 100 ml 100 to ml 100 to

, 30 ml) ampholytes ®

O O O O O 2 2 2 2 2 -mercaptoethanol ml) DTT or 3% (0.4 immediately catalog #161-0737 Tris base Tris SDSSample Solubilization Buffer (50 ml) (pH mM Tris-HCl SDS, 9.5) 100 (w/v) 1% SDS diH DTT diH Bio-Lyte CHAPS withTitrate pH 9.5 diluted to HCl. diH 2% (v/v) carrier (v/v) 2% ampholytes (pH 3–10) Urea Thiourea diH Store as 1–2 ml andStore aliquots as 1–2 add at –70°C b use. before ml) (100 Solution Precipitation Protein Trichloroacetic20% DTT acid 0.2% in ice-cold (TCA), acetone (–20°C) TCA DTT Acetone Acetone Store at –20°C. ml) (100 Solution Wash DTT0.2% in ice-cold acetone (–20°C) DTT Acetone Acetone Store at –20°C. LysisBuffer (50 ml) DTT, (w/v) CHAPS, (w/v) 1% 4% M urea,M thiourea,7 2 Dissolve Dissolve SDS-PAGE Sample Buffer(2x, 8 ml) ( 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, SDS, 2% pH 25% 6.8, mM Tris-HCl, 62.5 blue bromophenol 0.01% pH 6.8 mM Tris-HCl, 0.5 25% Glycerol25% blue Bromophenol 1.0% diH 10% SDS 10%

1 g g 1

80 ml 80 ml to 1 L to 0.15 g 0.15 0.10 g 0.10 2.5 ml 2.5 1.44 g 1.00 g 1.00 0.24 g 0.20 g 0.20 0.88 g 0.88 6.06 g 8.00 g 800 ml 12.11 g 12.11 ~60 ml 100 mg 100 to 10 ml 10 to ml 10 to to 100 ml 100 to to 100 ml 100 to ml 100 to

O) 2 O (diH 2

4

4

O O O O O O O O O PO HPO 2 2 2 2 2 2 2 2 2 2 2

KH Deionized H Deionized Sample Preparation Buffers Sample Preparation 1 M Tris-HCl, (100 ml) pH 7.6 base Tris Buffer Formulations diH Adjust pH to 7.6 with HCl. Adjust 7.6 pH to diH KCl Na pH 7.6 1 M Tris-HCl, diH L) 1 (PBS, Buffered Saline Phosphate sodium (w/v) chloride0.9% mM phosphate in 10 buffer, pH 7.4 NaCl with HCl or NaOH. Adjust 7.4 pH to diH Adjust pH with 6.8 to HCl. Bromophenol1.0% Blue ml) (10 SolubilizationRIPA Buffer (100 ml) 0.5 M Tris-HCl, pH 6.8 (100 ml) (catalog #161-0799) base Tris diH diH Store at 4°C. SDS ml)10% (10 (catalog #161-0416) SDS #161-0404) (catalog blue Bromophenol diH mM NaCl, 5 mM EDTA, 150 pH mM 7.6, Tris-HCl 25 sodium deoxycholate, 1% X-100, Triton NP-40 or 1% 1% SDS 0.1% NaCl diH EDTA X-100 NP-40 or Triton deoxycholate Sodium SDS diH

64 Electrophoresis Guide

TABLE OF CONTENTS 67 Methods

to 1 L to 25.00 g 25.00 100 mg 100 60.60 g to 10 ml 10 to ~900ml to 250 ml 250 to O O O O 2 2 2 2 1.0% Bromophenol1.0% Blue ml) (10 #161-0404) (catalog blue Bromophenol diH Adjust pH with 6.8 to HCl. diH Buffer Components Store at 4°C. SDS10% (250 ml) (catalog #161-0416) SDS diH 0.5 M Tris-HCl, pH 6.8 L) (1 (catalog #161-0799) base Tris diH to 1 L to to 1 L to 1 L to 10.00 g 10.00 10.00 g 10.00 30.30 g 30.30 g 121.10 g 121.10 144.10 g 144.10 144.10 g 144.10 179.20 g 179.20

O O O 2 2 2

(catalog #161-0732)pH 8.3 SDS, 1% glycine, M 1.92 Tris, mM 250 base Tris SDS diH Running Buffers 10x SDS-PAGE L) (1 Glycine Buffer Formulations Do not adjust the pH (~pH 8.3). Do not adjust 8.3). the pH (~pH Do not adjust the pH (~pH 8.3). Do not adjust 8.3). the pH (~pH 10x Native L) (1 PAGE (catalog #161-0734) M glycine, pH 8.3 1.92 mM Tris, 250 base Tris diH Do not adjust 8.3). the pH (~pH L) (1 10x Tris-Tricine (catalog #161-0744) SDS, pH 8.3 1% 1 M Tricine, 1 M Tris, base Tris Glycine Tricine SDS diH

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69 69 Troubleshooting

Part III Troubleshooting is a straightforward Electrophoresis may problems However, technique. occasionally arise during various the workflow. electrophoresis in the steps This potential section highlights and causes, their and problems solutions. potential provides

68

68 Electrophoresis Guide

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71

Troubleshooting Figure 1 provides an example an of optimal gel image, where the resolved, nicely are bands each lane is very straight, and protein bands are present across the length the of gel (there is excellent separation across the entire range). molecular weight Fig. 1. Schematic of protein protein of Schematic 1. Fig. SDS-PAGE. during migration TIPS

fits undertopof at gasketnotch Ensure that edge top short of plate Ensure that top of short plate gasket green the touches Solution Solution not remove pipet tip from well before last sample of has left tip as it may bounce off bottom or sides and flow nextinto well. Do not squirt sample quickly into well, well, squirt quickly sample into not 25% each 25% IncreaseAPS and TEMED by degas50%; Use different %T reagents electrophoresis-grade Use Use correct %C Check solutions or weights Use correct %C glycerolInclude in 10% sample make it to denser buffer surrounding than Do slowly. well into sample Pipet ensureto wells are completely covered connections and electrodes Check Check buffer composition and type Check buffer protocol and concentrate if necessary Decrease voltage 25–50% by Check buffer composition and type Desalt sample gasketWet with running buffer Reduce APS and TEMED by Remove tape • • Fill buffer chamber with running buffer buffer outer chambers and inner Fill use before

polymerization min time <10 Gel inhibition; polymerization hr time >2 %T Low Poor quality acrylamide or bis little cross-linkerToo Bis concentration high too Cross-linker high too Sample not dense enough Pipetting, loading error gel cassette not removed buffer chamber disconnection Electrical Running buffer concentrated too and gel temperature high; too incorrect running buffer concentration or type used Running or reservoir buffer dilutetoo Voltage high too Incorrect running buffer composition or type sample in salt Excessive Incomplete gasket seal Excess catalysts; at the bottom precast of Tape the electrode/companion assembly Insufficient buffer in inner buffer chamber Insufficient bufferouter in Improper assembly gel of into

Cause Cause Gel feels soft Gel turns white Gel brittle Sample floats outwellof expected, and samples do not gel into migrate Gels run faster than expected Gels run slower than expected Buffer leaking from chamberinner Problem Swirls in gel Problem Current zero or less than

Electrophoresis Electrophoresis

min 2-D cleanup kit) to 2-D ™ protein solubilization waves during cell lysis and diminish carbohydrate content carbohydrate diminish Fragment DNA with ultrasonic ultrasonic with DNA Fragment endonucleases Add benzonase) example (for TCA/acetone with protein Precipitate Prepare fresh catalyst solution Increase catalyst concentration Prepare fresh catalyst solution Increase catalyst concentration Wash gasket if it is dirty Replace flawed wornor out casting stand gaskets stackingof APS gel 0.06% to TEMED and 0.12% stackingof APS gel 0.06% to and 0.12% TEMED and 0.12%

(ReadyPrep Solution Solution Ensure plates are free flaws of Ensure plates are aligned correctly Add Tris base untilAdd buffer Tris turns

Cast at room temperature, necessary if plates glass warming reagents electrophoresis-grade Use Prepare fresh APS prior casting to stacking gel • • immediately solution monomer Degas • • 10–15 solutions monomer Degas • • • • •

Use 0.05% APS and 0.05% TEMED APS and 0.05% Use0.05% Rinse or wipe off powder residue before each use blue again

Chipped glass plates Spacer plate and short plate not level Casting stand gasket dirty, Sample buffer is acidic too

Temperature too low too Temperature Poor quality acrylamide or bis Old APS (oxygen inhibits polymerization) Incorrect catalyst concentration

flawed, wornor out Incorrect catalyst used degassed not solution Monomer Failure degas to Too little or muchToo too APS or TEMED Powder residue has built up at pivot point pressure of cams High DNA or carbohydrate content

Cause Cause

yellow Problem Leaking during handcasting Laemmli sample buffer turns Problem

Webbing; excess acrylamide behind the comb Poor well formation

Pressure cams on casting frame are difficultto close or make noise when closed Gel does not polymerize Sample very viscous

Gel Casting and Sample Loading Sample and Casting Gel Sample Preparation

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TABLE OF CONTENTS 73 Troubleshooting

diluting stock or 10x 5x thorough mixing, especially mixing, when thorough 4.5% or 5% T or 5% 4.5% acrylamide residual remove Increase stacking of %T gel to Increase during current 25% by rate polymerization Decrease carefully gels Overlay Rinse wells after removing comb to Use electrophoresis-grade reagents reagents electrophoresis-grade Use conditions polymerization Check buffer bufferCheck composition; not mixed well, or buffer in upper chamber concentrated too ensuring buffer, new Prepare Minimize salts, detergents, and solvents in sample preparation buffers sample and voltage correct Use Remove salts from sample by Use same buffer in samples as in gel and bufferCheck composition instructions dilution to sampleto preparation sample between time Minimize startup power and application power setting V from 200 150 V to or fill lower chamberto within 1 cm short of top of plate buffer outer and inner Fill chambers ensure to that wells are completely covered less protein Load prior column desalting or dialysis Do not exceed recommended power Decrease conditions. running

Solution Prepare fresh solutions Use external cooling during run or slowly more run stacking • • • • • • • • •

Sample preparation/buffer issues conditions running Incorrect Excess salt in samples Ionic strength sample of lower than that gel of buffer wrong Insufficient sample or formulation current on turning Diffusion to prior through Diffusion migration during

Insufficient buffer Overloaded proteins Power conditions excessive bis-acrylamide, incomplete incomplete bis-acrylamide, Poor quality acrylamidePoor or Old SDS or sample buffer Gel temperature high too Excess heating gel; of center of stacking gel Uneven gel interface gel runs hotter than either end

Cause gel lane Skewed or distorted bands, spreading band lateral

within bands frowning or Smiling

Problem bands Diffuse broad or polymerization Bands “smile” across gel, band pattern curves upward at both sides gel of Evaluation of Separationof Evaluation

Restrict duration incubation of Wash gel in water or respective thoroughly trays Clean staining Limit time that gels and staining handle and gloves dust-free Use Ensure that U-shaped electrode core gasket is clean, free cuts, of and lubricated with buffer Ensure that short plate is gasket on notch under in staining solutions as protocol in recommended destaining solution for min ≥30 solution are exposed open to air gels only edges by volume (appropriate to gel size) gel to (appropriate volume time staining Increase Repeat staining protocol with fresh solution staining Solution Solution troubleshooting, or contact manufacturer instrument imaging manual instrument Check Clean staining trays and other laboratory with equipment cleaner glassware stain for recommendations Follow confirm there is protein for manual instrument Check Clean staining trays and other laboratory with equipment cleaner glassware Stain with another method to spacer plate • • • • • • • Use high-purity water and reagents staining for Keep buffer level below of top Agitate gel during staining gel during Agitate gel water forTransfer rehydration to

Insufficienttime staining Reuse staining of solution were parameters imaging Incorrect Dirty staining trays volume Insufficientstain malfunctioning system Imaging Dirty equipment or staining trays much time in stainingToo solution No protein in gel Improper assembly Improper Reagent impurities Particulate material from reagents, Upper buffer chamber overfilled staining dust, or gloves tray, Insufficient shakingstaining during Gel dehydrated

Tetra cell) Tetra (contd.) ®

Cause Cause

background uneven or High

used Poor staining sensitivity staining

Leaking from upper buffer chamber buffer upper from Leaking Problem Bands not visible Problem

Speckles or blotches in gel image staining Uneven (Mini-PROTEAN Gel shrinkage Total Protein Staining Protein Total Electrophoresis Electrophoresis

72 Electrophoresis Guide

TABLE OF CONTENTS 75 Troubleshooting Dilute sampleDilute predominant remove Selectively protein in sample (fractionate) Reduce minimize to voltage 25% by Centrifuge samples remove to loading sample to prior particulates Dilute sample in samplebuffer Solution • • • streaking • • DTT BMEUse or 1% 5% or a differentUse SDS-PAGE buffer system in native PAGE or IEF

Overloaded samples precipitation Sample Concentration reducingof agent low too Bands interest of may be neutral or positively charged in buffer used; pl bands of pH must units be ~2 more negative than pH of running buffer (contd.) Cause

Vertical streaking Problem bands down as expected Protein bands do not migrate Fuzzy or spurious artifactual Evaluation of Separationof Evaluation

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TABLE OF CONTENTS Electrophoresis Guide Appendices TABLE CONTENTS OF

Part IV Appendices

76 77 Electrophoresis Guide Appendices

Glossary Cathode Negatively charged electrode. Positively charged molecules (cations) move toward %C Cross-linker concentration; weight percentage of cross-linker in a polyacrylamide gel the cathode, which is usually colored black %T Monomer concentration (acrylamide + cross-linker) in a gel (in g/100 ml). Gels can CHAPS Zwitterionic detergent (having both positively and negatively charged groups be made with a single, continuous %T through the gel (single-percentage gels), or with a net charge of zero) that is widely used for protein solubilization for IEF and with a gradient of %T through the gel (gradient gels) 2-D electrophoresis; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

2-D electrophoresis Two-dimensional electrophoresis. Proteins are separated first according to Comb Object used to cast wells in an agarose or acrylamide gel. In PAGE applications, isoelectric point (pI) by isoelectric focusing (IEF) and then according to size by square-bottom combs are inserted into the gel sandwich before polymerization to SDS-PAGE, yielding a two-dimensional protein map of spots form square-bottomed wells

2-Mercaptoethanol Reducing agent necessary for cleavage of intra- and inter-molecular disulfide bonds Continuous buffer Gel-based electrophoresis system that uses the same buffer (at constant pH) in to achieve complete protein unfolding and to maintain proteins in a fully reduced system the gel, sample, and electrode reservoirs. Typically a single-percentage gel is used, state. Also known as b-mercaptoethanol or BME and the sample is loaded directly into the part of the gel in which separation occurs. Continuous systems are not common in protein separations; they are used mostly Acrylamide Monomer used with a cross-linker to form the matrix used for separating proteins or for nucleic acid analysis small DNA molecules Coomassie Anionic dye used in the total protein staining of gels and blots that comes in two Ammonium persulfate Initiator used with TEMED (catalyst) to initiate the polymerization of acrylamide and (Brilliant) Blue forms: Coomassie (Brilliant) Blue G-250 differs from Coomassie (Brilliant) Blue (APS) bisacrylamide in making a polyacrylamide gel; (NH ) S O 4 2 2 8 R-250 by the addition of two methyl groups Ampholyte Amphoteric molecule (containing both acidic and basic groups) that exists mostly Criterion™ cells, Family of Bio-Rad products used for midi-format vertical electrophoresis; as a zwitterion in a certain pH range. Ampholytes are used to establish a stable pH blotters, and gels includes the Criterion and Criterion™ Dodeca™ cells, Criterion blotter, and Criterion gradient for use in isoelectric focusing precast gels Anode Positively charged electrode. Negatively charged molecules (anions) move toward TABLE CONTENTS OF Cross-linker Molecule (for example, bis-acrylamide) used to link polymerizing monomer the anode, which is usually colored red molecules together to form the gel, a netlike structure. Holes in the nets are called Anionic dye Negatively charged compound used as a stain; used in blotting to stain proteins pores, and pore size is determined in part by the cross-linker concentration. Pores immobilized on membranes such as nitrocellulose or PVDF may or may not sieve the macromolecules

Antibody Immunoglobulin; protein produced in response to an , which specifically DC™ assay kit Bio-Rad’s detergent-compatible protein assay kit binds the portion of the antigen that initiated its production Discontinuous Gel-based electrophoresis system that uses different buffers and sometimes Antigen Foreign molecule that specifically binds with an antibody buffer system different buffer compositions to focus and separate components of a sample. Discontinuous systems typically focus the proteins into tighter bands than Assay Analysis of the quantity or characteristics of a substance continuous gel systems, allowing larger protein loads Background Nonspecific signal or noise that can interfere with the interpretation of valid signals Dithiothreitol (DTT) Reducing agent necessary for cleavage of intra- and inter-molecular disulfide Bio-Spin® columns Family of Bio-Rad sample preparation products that includes the Bio-Spin 6 and bonds to achieve complete protein unfolding and to maintain all proteins in a fully Micro Bio-Spin™ 6 columns used for buffer exchange and desalting applications reduced state

Bis or bis-acrylamide A common cross-linker used with acrylamide to form a support matrix; Electroelution Technique that applies the principles of electrophoresis to enable recovery (elution) N,N'-methylene-bis-acrylamide of molecules such as proteins from gels and gel slices

Blocking reagent Protein used to saturate unoccupied binding sites on a blot to prevent nonspecific Electrophoresis Movement of charged molecules in a uniform electric field binding of antibody or protein probes to the membrane Glycerol Small nonionic molecule used in vertical gel electrophoresis to increase the density Blot Immobilization of proteins or other molecules onto a membrane, or a membrane that of the sample buffer so that it sinks to the bottom of the sample well; also used to has the molecules adsorbed onto its surface help keep proteins soluble, especially in isoelectric focusing

Blue native PAGE Discontinuous electrophoretic system that allows high-resolution separation of Glycine  used as the trailing ion in discontinuous electrophoresis complexes in native, enzymatically active states. Membrane Gradient gel Gel with gradually changing monomer concentration (%T) in the direction of protein complexes are solubilized by neutral, nondenaturing detergents like migration. In SDS-PAGE, gradients are used to separate wider molecular weight n-dodecyl-b-D-maltoside. After addition of Coomassie (Brilliant) Blue G-250, which ranges than can be separated with single-percentage gels binds to the surface of the proteins, separation of the negatively charged complexes according to mass is possible Immobilized pH Strips in which buffering groups are covalently bound to an acrylamide gel matrix, gradient (IPG) resulting in stable pH gradients except the most alkaline (>12) pH values. This Bromophenol blue Common tracking dye used to monitor the progress of electrophoresis eliminates problems of gradient instability and poor sample loading capacity Carrier ampholytes Heterogeneous mixture of small (300–1,000 Da) polyamino-polycarboxylate buffering associated with carrier ampholyte–generated pH gradients compounds that have closely spaced pI values and high conductivity. Within an electric field, they align according to pI to establish the pH

78 79 Electrophoresis Guide Appendices

Immunoassay Test for a substance by its reactivity with an antibody Protein standards Mixtures of well-characterized or recombinant proteins used to monitor separation and estimate the size and concentration of the proteins separated in a gel Immunoblotting Blot detection by antibody binding Prestained standards Mixture of molecular weight marker proteins that have covalently attached dye Immunodetection Detection of a molecule by its binding to an antibody molecules, which render the bands visible during electrophoresis and transfer Immunoglobulin Antibody; protein produced in response to an antigen, which specifically binds the RC DC™ assay kit Bio-Rad’s reductant- and detergent-compatible protein assay kit portion of the antigen that initiated its production Resolving gel Portion of a discontinuous electrophoresis gel that separates the different bands Ion front Group of ions moving together during electrophoresis, marking the movement of the from each other buffer from the upper buffer reservoir. Due to their small size, they are not hindered

by a sieving matrix and move together primarily because of their charge Rf value Relative distance a protein has traveled compared to the distance traveled by the ion front. This value is used to compare proteins in different lanes and even in different Ionic strength Measure of the ionic concentration of a solution that affects its resistance gels. It can be used with standards to generate standard curves, from which the Isoelectric focusing Electrophoresis technique that separates proteins according to their isoelectric molecular weight or isoelectric point of an unknown may be determined (IEF) point (pI) Running buffer Buffer that provides the ions for the electrical current in an electrophoresis run. Isoelectric point (pI) pH value at which a molecule carries no electrical charge, or at which the negative It may also contain denaturing agents. The running buffer provides the trailing ions in and positive charges are equal discontinuous electrophoresis

Ligand Molecule that binds another in a complex Sample buffer Buffer in which a sample is suspended prior to loading onto a gel. SDS-PAGE sample buffer typically contains denaturing agents (including reducing agents and MicroRotofor™ Family of Bio-Rad sample preparation products, including the MicroRotofor SDS), tracking dye, and glycerol cells and kits liquid-phase IEF cell and MicroRotofor cell lysis kits SDS-PAGE Separation of molecules by molecular weight in a polyacrylamide gel matrix in the Monomer Unit that makes up a polymer (acrylamide is a monomer that is polymerized into presence of a denaturing detergent, (SDS). SDS denatures TABLE CONTENTS OF polyacrylamide) polypeptides and binds to proteins at a constant charge-to-mass-ratio. In a sieving Mini-PROTEAN® Family of Bio-Rad products used for mini-format vertical electrophoresis; polyacrylamide gel, the rate at which the resulting SDS-coated proteins migrate in cells and gels includes the Mini-PROTEAN Tetra and Mini-PROTEAN® 3 Dodeca™ cells, and the gel is relative only to their size and not to their charge or shape Mini-PROTEAN precast gels Secondary antibody Reporter antibody that binds to a primary antibody; used to facilitate detection Native PAGE Version of PAGE that retains native protein configuration, performed in the absence Sodium dodecyl Anionic detergent that denatures proteins and binds to polypeptides in a constant of SDS and other denaturing agents sulfate (SDS) weight ratio of 1:4 (SDS:polypeptide) Ohm’s Law Describes the mutual dependence of three electrical parameters (V, volts; Spacers Small blocks set between the two glass plates at the sides of a gel cassette, I, ampere; R, ohm): V = I x R which create a space between the glass plates in which to pour the slab gel PAGE Polyacrylamide gel electrophoresis, a common method of separating proteins monomer solution

Polyacrylamide Anticonvective sieving matrix used in gel electrophoresis. Polyacrylamide gels are Stacking gel Portion of a discontinuous electrophoresis gel that concentrates the components of cast using mixtures of acrylamide monomers with a cross-linking reagent, usually the sample to create a very thin starting zone; bands are then separated from each N,N'-methylenebisacrylamide (bis), both solubilized in buffer other in the resolving gel

Polyacrylamide gel Electrophoresis technique that uses polyacrylamide as the separation medium Stain-free technology Protein detection technology involving UV-induced haloalkane modification of protein electrophoresis (PAGE) tryptophan residues. Continued exposure to UV light causes fluorescence of the modified proteins, which are then detected by a CCD imager. Sensitivity of this PowerPac™ Family of Bio-Rad power supplies technique is generally equal to or better than Coomassie staining power supplies Stained standards Mixture of molecular weight marker proteins that have covalently attached dye Power supply Instrument that provides the electric power to drive electrophoresis and molecules; the bands are visible during electrophoresis and transfer electrophoretic blotting experiments Standard Collection of molecules with known properties, such as molecular weight or Precision Plus Protein™ Bio-Rad’s family of recombinant protein standards isoelectric point. Often used to create standard curves, from which properties of an standards unknown may be determined Preparative Electrophoresis techniques that separate large volumes of protein samples TEMED Used with APS (initiator) to catalyze the polymerization of acrylamide and electrophoresis (nanogram to gram quantities of protein), generally for the purposes of purification bisacrylamide in making a polyacrylamide gel; N,N,N',N'-tetramethylethylenediamine or fractionation (to reduce sample complexity) TGX™ gels Bio-Rad’s Tris-glycine extended shelf life precast gels Primary antibody Antibody that binds a molecule of interest PROTEAN® cells Family of Bio-Rad products used for large-format vertical electrophoresis; includes the PROTEAN II xi, PROTEAN II XL, and PROTEAN Plus® Dodeca™ cells

80 81 Electrophoresis Guide Appendices

Total protein stain Reagent that binds nonspecifically to proteins; used to detect the entire protein References and Related Reading pattern on a blot or gel References Sample preparation and protein assay Tricine Organic compound used in SDS-PAGE as a buffer component to replace glycine Berkelman T (2008). Quantitation of protein in samples prepared for 2-D electrophoresis. Methods Mol Biol 424, 43–49. and improve resolution of small (down to 1–5 kD) proteins Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. Tris Organic component of buffer solutions that has an effective pH range of 7.0–9.2; Cañas B et al. (2007). Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153, 235–258. tris(hydroxymethyl) aminomethane Chen Y et al. (2008). Sample preparation. J Chromatogr A 1184, 191–219. Transfer Immobilization of proteins or other molecules onto a membrane by electrophoretic Damerval C et al. (1988). Two-dimensional electrophoresis in plant biology. Advances in Electrophoresis 2, 236–340. or passive means Drews O et al. (2004). Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis. Proteomics 4, 1293–1304. Triton X-100 Nonionic detergent widely used for protein solubilization (for IEF and 2-D Evans DR et al. (2009). Concentration of proteins and removal of solutes. Methods Enzymol 463, 97–120. electrophoresis) Goldberg S (2008). Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol Biol 424, 3–22. Tween 20 Nonionic detergent; used in blot detection procedures as a blocking reagent or in Harder A et al. (1999). Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Electrophoresis 20, 826–829. wash buffers to minimize nonspecific binding and background Huber LA et al. (2003). Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res. 92, 962–968. Unstained standards Mixture of molecular weight marker proteins that do not have covalently attached Lowry OH et al. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. dye molecules; the bands are invisible during electrophoresis and transfer, but are Luche S et al. (2003). Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. useful for molecular weight determination Proteomics 3, 249–253. Noble JE and Bailey MJ (2009). Quantitation of protein. Methods Enzymol 463, 73–95. Urea Chaotrope usually included at rather high concentrations (9.5 M) in sample Poetsch A and Wolters D (2008). Bacterial membrane proteomics. Proteomics 8, 4100–4122. solubilization buffers for denaturing IEF and 2-D PAGE Posch A et al. (2006). Tools for sample preparation and prefractionation in two-dimensional gel electrophoresis. In Separation Methods in Proteomics, Smejkal GB ed. (Boca Raton: CRC Press), 107–133. Western blotting Immobilization of proteins onto a membrane and subsequent detection by Rabilloud T (1996). Solubilization of proteins for electrophoretic analyses. Electrophoresis 17, 813–829. TABLE CONTENTS OF protein-specific binding and detection reagents Rhodes DG and Laue TM (2009). Determination of protein purity. Methods Enzymol 463, 677–689. Zymogram PAGE Electrophoresis technique used to detect and characterize collagenases and Sapan CV et al. (1999). Colorimetric protein assay techniques. Biotechnol Appl Biochem 29, 99–108. other proteases within the gel. Gels are cast with gelatin or casein, which Smith PK et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85. acts as a substrate for the enzymes that are separated in the gel under Vuillard L et al. (1995). Enhancing protein solubilization with nondetergent sulfobetaines. Electrophoresis 16, 295-297. nonreducing conditions Electrophoresis Andrews AT (1986). Electrophoresis: theory, techniques and biochemical and clinical applications (New York: Oxford University Press). Bjellqvist B et al. (1982). Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods 6, 317–339. Davis BJ (1964). Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 121, 404–427. Dunn MJ (1993). Gel electrophoresis: Proteins (Oxford: BIOS Scientific Publishers Ltd.). Fenselau C (2007). A review of quantitative methods for proteomic studies. J Chromatogr B Analyt Technol Biomed Life Sci 855, 14–20. Garfin DE (1990). One-dimensional gel electrophoresis. Methods Enzymol 182, 425–441. Garfin DE (2009). One-dimensional gel electrophoresis. Methods Enzymol 463, 497–513. Goldenberg DP and Creighton TE (1984). Gel electrophoresis in studies of protein conformation and folding. Anal Biochem 138, 1–18. Hames BD (1998). Gel electrophoresis of proteins: A practical approach, 3rd ed. (Oxford: Oxford University Press). Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. McLellan T (1982). Electrophoresis buffers for polyacrylamide gels at various pH. Anal Biochem 126, 94–99. Niepmann M (2007). Discontinuous native protein gel electrophoresis: pros and cons. Expert Rev Proteomics 4, 355–361. Nijtmans LG et al. (2002). Blue Native electrophoresis to study mitochondrial and other protein complexes. Methods 26, 327–334. O'Farrell PH (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007–4021. Ornstein L (1964). Disc electrophoresis I: background and theory. Ann NY Acad Sci 121, 321–349. Rabilloud T (2010). Variations on a theme: changes to electrophoretic separations that can make a difference. J Proteomics 73, 1562–1572. Reisinger V and Eichacker LA (2008). Isolation of membrane protein complexes by blue native electrophoresis. Methods Mol Biol 424, 423–431. Schägger H and von Jagow G (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379. Vavricka SR et al. (2009). Serum protein electrophoresis: an underused but very useful test. Digestion 79, 203–210. Westermeier R (2004). Isoelectric focusing. Methods Mol Biol 244, 225–232. Wheeler D et al. (2004). Discontinuous buffer systems operative at pH 2.5 - 11.0, 0 degrees C and 25 degrees C, available on the Internet. Electrophoresis 25, 973–974. Zewert TE and Harrington MG (1993). Protein electrophoresis. Curr Opin Biotechnol 4, 3–8.

82 83 Electrophoresis Guide Appendices

Staining Product Information Agrawal GK and Thelen JJ (2009). A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein Bulletin Title identification and quantitative profiling. Methods Mol Biol 527, 3–19. 5517 MicroRotofor™ Lysis Kits Flier Fernandez-Patron C et al. (1992). Reverse staining of sodium dodecyl sulfate polyacrylamide gels by -zinc salts: sensitive detection of 1069 Colorimetric Protein Assays unmodified proteins. Biotechniques 12, 564-573. 2826 SmartSpec™ Plus Spectrophotometer Brochure Gottlieb M and Chavko M (1987). Silver staining of native and denatured eucaryotic DNA in agarose gels. Anal Biochem 165, 33-37. 2414 The Little Book of Standards Hart C et al. (2003). Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24, 588–598. 2998 Protein Standards Application Guide Lee C et al. (1987). Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 166, 308-312. 2317 Ready-to-Run Buffers and Solutions Brochure ® Merril CR (1987). Detection of proteins separated by electrophoresis. Adv Electrophoresis 1, 111–139. 5535 Mini-PROTEAN Tetra Cell Brochure ® ™ Merril CR et al. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 5871 Mini-PROTEAN TGX Precast Gels Product Information Sheet 211, 1437–1438. 2710 Criterion™ Precast Gel System Brochure Miller I et al. (2006). Protein stains for proteomic applications: which, when, why? Proteomics, 6, 5385–5408. 2911 Criterion XT Precast Gels Product Information Sheet Neuhoff V et al. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at 5974 Criterion TGX Stain-Free Precast Gels Product Information Sheet nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262. 1760 PROTEAN ® II xi and XL Cells Product Information Sheet Oakley BR et al. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105, 361–363. 2881 PowerPac™ Basic 300 V Power Supply Flier Rabilloud T et al. (1994). Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40, 57–75. 2882 PowerPac HC High-Current Power Supply Flier Simpson RJ (2010). Rapid coomassie blue staining of protein gels. Cold Spring Harb Protoc, pdb prot5413. 2885 PowerPac Universal Power Supply Brochure Sinha P et al. (2001). A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of 3189 PowerPac HV Power Supply Brochure proteins after two-dimensional electrophoresis. Proteomics 1, 835-840. 2423 Bio-Safe™ Coomassie Stain Brochure Steinberg TH (2009). Protein gel staining methods: an introduction and overview. Methods Enzymol 463, 541–563. 5346 Flamingo™ Fluorescent Gel Stain Product Information Sheet Steinberg TH et al. (2003). Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3, 1128–1144. 5900 Oriole™ Fluorescent Gel Stain Product Information Sheet Westermeier R and Marouga R (2005). Protein detection methods in proteomics research. Biosci Rep 25, 19–32. 5888 Bio-Rad Imaging Systems Family Brochure Yan JX et al. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization

TABLE CONTENTS OF and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672. 3096 Expression Proteomics Brochure 2032 Western Blotting Detection Reagents Brochure Bio-Rad Bulletins 5294 MicroRotofor Cell Product Information Sheet Technical Notes Bulletin Title Instruction Manuals 2651 2-D Electrophoresis for Proteomics: A Methods and Product Manual Bulletin Title 2895 Protein Blotting Guide, A Guide to Transfer and Detection 10007296 Mini-PROTEAN Tetra Cell 1156 Acrylamide Polymerization – a Practical Approach 1658100 Mini-PROTEAN Precast Gels ™ 1909 Modification of Bio-Rad DC Protein Assay for Use with Thiols 4006183 Criterion Cell ™ ™ 6001 Rapid Validation of Purified Proteins Using Criterion Stain Free Gels 4006213 PowerPac Basic Power Supply ® ™ 5910 Mini-PROTEAN TGX Precast Gel: A Gel for SDS-PAGE with Improved Stability — Comparison with Standard Laemmli Gels 4006222 PowerPac HC Power Supply 5911 Mini-PROTEAN TGX Precast Gel: A Versatile and Robust Laemmli-Like Precast Gel for SDS-PAGE 4006223 PowerPac Universal Power Supply ® 5932 Ready Gel to Mini-PROTEAN TGX Precast Gels Catalog Number Conversion Chart 4110001 Criterion Gel Application Guide 5934 NuPAGE Bis-Tris Precast Gels (MOPS Buffer) to Mini-PROTEAN TGX Precast Gels Catalog Number Conversion Chart 4110130 Criterion XT Precast Gels 3133 Molecular Weight Determination by SDS-PAGE 4110065 Quick Start™ Bradford Protein Assay ™ 3144 Using Precision Plus Protein Standards to Determine Molecular Weight 4110107 RC DC™ Protein Assay 5956 Precision Plus Protein Dual Xtra Standards – New Protein Standards with an Extended Range from 2 to 250 kD 4000126-14 The Discovery Series: Quantity One® 1-D Analysis Software ™ ™ ™ 5763 Molecular Weight Estimation Using Precision Plus Protein WesternC Standards on Criterion Tris-HCl and Criterion XT Bis-Tris Gels LIT33 Bio-Rad Protein Assay 5576 Molecular Weight Estimation and Quantitation of Protein Samples Using Precision Plus Protein WesternC Standards, LIT448 DC Protein Assay the Immun-Star™ WesternC™ Chemiluminescent Detection Kit, and the Molecular Imager® ChemiDoc™ XRS Imaging System 2043 Purification of Proteins from Mycobacterium tuberculosis 2168 Isolation of Hydrophobic C. albicans Cell Wall Protein by In-Line Transfer From Continuous Elution Preparative Electrophoresis 2376 Gel Electrophoresis: Separation of Native Basic Proteins by Cathodic, Discontinuous Polyacrylamide Gel Electrophoresis 5705 Sensitivity and Protein-to-Protein Consistency of Flamingo™ Fluorescent Gel Stain Compared to Other Fluorescent Stains 5754 Comparison of SYPRO Ruby and Flamingo Fluorescent Gel Stains with Respect to Compatibility with Mass Spectrometry 5921 Oriole™ Fluorescent Gel Stain: Characterization and Comparison with SYPRO Ruby Gel Stain 5989 Imaging Fluorescently Stained Gels with Image lab™ Software 5723 Increase Western Blot Throughput with Multiplex Fluorescent Detection 5294 MicroRotofor Cell Product Information Sheet

84 85 Electrophoresis Guide Appendices

Ordering Information Catalog # Description Catalog # Description Catalog # Description Electrophoresis Instrumentation 165-8033 Mini-PROTEAN Tetra Cell, Mini Trans-Blot PROTEAN® Plus Dodeca™ Cells and Systems Protein Assay Kits and Instruments Catalog # Description Module, and PowerPac Basic Power Supply, 165-4150 PROTEAN Plus Dodeca Cell, 100/120 V, includes 500-0001 Bio-Rad Protein Assay Kit I, includes 450 ml dye includes 165-8001, 170-3935, and 164-5050 electrophoresis buffer tank with built-in ceramic reagent concentrate, bovine b-globulin standard; Mini-PROTEAN® Tetra Cells and Systems 165-8034 Mini-PROTEAN Tetra Cell for Mini Precast Gels, cooling core, lid, buffer recirculation pump with sufficient for 440 standard assays or 2,200 165-8000 Mini-PROTEAN Tetra Cell, 10-well, 0.75 mm Mini Trans-Blot Module, and PowerPac Basic tubing, 2 gel releasers microplate assays thickness; 4-gel system includes 5 combs, 5 sets Power Supply, includes 165-8004, 170-3935, 165-4140 PROTEAN Plus Dodeca Cell (100/120 V) and 500-0002 Bio-Rad Protein Assay Kit II, includes 450 ml of glass plates, 2 casting stands, 4 casting frames, and 164-5050 PowerPac HC Power Supply, includes 165-4150 dye reagent concentrate, bovine serum albumin sample loading guide, electrode assembly, and 164-5052 standard; sufficient for 440 standard assays or companion running module, tank, lid with power 165-8035 Mini-PROTEAN Tetra Cell, Mini Trans-Blot 2,200 microplate assays cables, mini cell buffer dam Module, and PowerPac HC Power Supply, 165-4142 PROTEAN Plus Dodeca Cell (100/120 V) and includes 165-8001, 170-3935, and 164-5052 PowerPac Universal Power Supply, includes 500-0111 DC™ Protein Assay Kit I, includes 250 ml alkaline 165-8001 Mini-PROTEAN Tetra Cell, 10-well, 1.0 mm 165-4150 and 164-5070 copper tartrate solution, 2 L dilute Folin reagent, thickness; 4-gel system includes 5 combs, 5 sets 165-8036 Mini-PROTEAN Tetra Cell for Mini Precast Gels, 5 ml surfactant solution, bovine -globulin standard; of glass plates, 2 casting stands, 4 casting frames, Mini Trans-Blot Module, and PowerPac HC Power 165-4144 PROTEAN Plus Dodeca Cell (100/120 V), b sufficient for 450 standard assays sample loading guide, electrode assembly, Supply, includes 165-8004, 170-3935, and 164-5052 Trans-Blot Plus Cell, and PowerPac Universal Power companion running module, tank, lid with power Mini-PROTEAN® Dodeca™ Cells and Systems Supply, includes 165-4150, 170-3990, and 164-5070 500-0112 DC Protein Assay Kit II, includes 250 ml alkaline cables, mini cell buffer dam 165-4100 Mini-PROTEAN 3 Dodeca Cell, includes 165-5134 PROTEAN Plus Dodeca Cell (100/120 V) and copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution, bovine serum albumin standard; 165-8002 Mini-PROTEAN Tetra Cell, 2 10-well, 0.75 mm electrophoresis tank with built-in cooling coil, lid with Two 6-Row AnyGel Stands, includes 165-4150 sufficient for 450 standard assays thickness; 2-gel system includes 5 combs, 5 sets of power cables, 6 electrophoresis clamping frames, and two 165-5131 ™ glass plates, casting stand, 2 casting frames, sample 2 buffer dams, drain line, 2 gel releasers 165-4151 PROTEAN Plus Dodeca Cell, 220/240 V, includes 500-0120 RC DC Protein Assay Reagents Package, loading guide, electrode assembly, tank, lid with 165-4101 Mini-PROTEAN 3 Dodeca Cell with Multi-Casting electrophoresis buffer tank with built-in ceramic includes 250 ml alkaline copper tartrate solution, power cables, mini cell buffer dam Chamber, same as 165-4100 with multi-casting cooling core, lid, buffer recirculation pump with 2 L dilute Folin reagent, 5 ml surfactant solution; sufficient for 450 standard assays 165-8003 Mini-PROTEAN Tetra Cell, 10-well, 1.0 mm chamber, 15 separation sheets, 8 acrylic blocks, tubing, 2 gel releasers thickness; 2-gel system includes 5 combs, 5 sets of tapered luer connector, stopcock valve 165-4141 PROTEAN Plus Dodeca Cell (220/240 V) and 500-0121 RC DC Protein Assay Kit I, includes RC reagents glass plates, casting stand, 2 casting frames, sample Criterion™ Cells and Systems PowerPac HC Power Supply, includes 165-4151 package, DC reagents package, bovine b-globulin loading guide, electrode assembly, tank, lid with 165-6001 Criterion Cell, includes buffer tank, lid with power and 164-5052 standard; sufficient for 450 standard assays power cables, mini cell buffer dam cables, 3 sample loading guides (12 + 2-well, 165-4143 PROTEAN Plus Dodeca Cell (220/240 V) and 500-0122 RC DC Protein Assay Kit II, includes RC reagents 165-8004 Mini-PROTEAN Tetra Cell for Mini Precast Gels, 18-well, 26-well) PowerPac Universal Power Supply, includes package, DC reagents package, bovine serum TABLE CONTENTS OF 4-gel system includes electrode assembly, clamping 165-6019 Criterion Cell and PowerPac Basic Power Supply, 165-4151 and 164-5070 albumin standard; sufficient for 450 standard assays frame, companion module, tank, lid with power ™ 100–120/220–240 V, includes 165-6001 and 164-5050 165-4145 PROTEAN Plus Dodeca Cell (220/240 V), 500-0201 Quick Start Bradford Protein Assay Kit 1, cables, mini cell buffer dam Criterion™ Dodeca™ Cells and Systems Trans-Blot Plus Cell, and PowerPac Universal Power includes 1x dye reagent (1 L), bovine serum albumin 165-8005 Mini-PROTEAN Tetra Cell for Mini Precast Gels, 165-4130 Criterion Dodeca Cell, includes electrophoresis Supply, includes 165-4151, 170-3990, and 164-5070 standard (5 x 2 mg/ml); sufficient for 200 standard 2-gel system includes electrode assembly, clamping assays or 4,000 microplate assays buffer tank with built-in cooling coil, lid with 165-5135 PROTEAN Plus Dodeca Cell (220/240 V) and frame, tank, lid with power cables, mini cell buffer dam power cables Two 6-Row AnyGel Stands, includes 165-4151 500-0202 Quick Start Bradford Protein Assay Kit 2, 165-8006 Mini-PROTEAN Tetra Cell, 10-well, 1.5 mm 165-4138 Criterion Dodeca Cell and PowerPac HC Power and two 165-5131 includes 1x dye reagent (1 L), bovine serum albumin thickness; 4-gel system includes 5 combs, 5 sets Supply, includes 165-4130 and 164-5052 standard set (2 sets of 7 concentration standards, of glass plates, 2 casting stands, 4 casting frames, Power Supplies 0.125–2.0 mg/ml, 2 ml) 165-4139 Criterion Dodeca Cell and PowerPac Universal 164-5050 PowerPac Basic Power Supply, 100–120/220–240 V sample loading guide, electrode assembly, 500-0203 Quick Start Bradford Protein Assay Kit 3, Power Supply, includes 165-4130 and 164-5070 164-5052 PowerPac HC Power Supply, 100–120/220–240 V companion running module, tank, lid with power includes 1x dye reagent (1 L), bovine b-globulin ™ cables, mini cell buffer dam 165-5133 Criterion Dodeca Cell and 6-Row AnyGel Stand, 164-5056 PowerPac HV Power Supply, 100–120/220–240 V standard (5 x 2 mg/ml) includes 165-4130 and 165-5131 164-5070 PowerPac Universal Power Supply, 165-8007 Mini-PROTEAN Tetra Cell, 10-well, 1.5 mm 500-0204 Quick Start Bradford Protein Assay Kit 4, ® 100–120/220–240 V thickness; 2-gel system includes 5 combs, 5 sets of PROTEAN II xi Cells includes 1x dye reagent (1 L), bovine b-globulin glass plates, casting stand, 2 casting frames, sample 165-1801 PROTEAN II xi Cell, 16 cm, without spacers and combs Sample Preparation Kits standard set (2 sets of 7 concentration standards, loading guide, electrode assembly, tank, lid with 165-1802 PROTEAN II xi Cell, 16 cm, 1.5 mm spacers (4), 163-2141 MicroRotofor™ Cell Lysis Kit (Mammal), 15 preps, 0.125–2.0 mg/ml, 2 ml) power cables, mini cell buffer dam 15-well combs (2) includes 50 ml protein solubilization buffer (PSB), ™ ™ 170-2525 SmartSpec Plus Spectrophotometer 165-8025 Mini-PROTEAN Tetra Cell and PowerPac™ Basic ReadyPrep mini grinders (2 packs of 10 each) 165-1803 PROTEAN II xi Cell, 16 cm, 1.0 mm spacers (4), 170-2502 Standard Cuvette, 1–3.5 ml, quartz Power Supply, includes 165-8001 and 164-5050 163-2142 MicroRotofor Cell Lysis Kit (Plant), 10 preps, 15-well combs (2) 170-2511 trUView™ Cuvettes, pack of 100, individually packaged, ™ includes 50 ml protein solubilization buffer (PSB), 165-8026 Mini-PROTEAN Tetra Cell and PowerPac 165-1804 PROTEAN II xi Cell, 16 cm, 0.75 mm spacers (4), disposable DNase- and RNase-free cuvettes Universal Power Supply, includes 165-8001 ReadyPrep 2-D cleanup kit (50 reaction size) 15-well combs (2) Sample Preparation Buffers and Reagents and 164-5070 163-2143 MicroRotofor Cell Lysis Kit (Yeast), 15 preps, 165-1811 PROTEAN II xi Cell, 20 cm, without spacers 161-0737 Laemmli Sample Buffer, 30 ml 165-8027 Mini-PROTEAN Tetra Cell and PowerPac™ HC includes 50 ml protein solubilization buffer (PSB), and combs 161-0738 Native Sample Buffer, 30 ml Power Supply, includes 165-8001 and 164-5052 15 ml yeast suspension buffer, 2 x 0.5 ml lyticase 165-1812 PROTEAN II xi Cell, 20 cm, 1.5 mm spacers (4), (1.5 U/µl) 161-0739 Tricine Sample Buffer, 30 ml 165-8028 Mini-PROTEAN Tetra Cell and PowerPac™ HV 15-well combs (2) 161-0763 IEF Sample Buffer, 30 ml Power Supply, includes 165-8001 and 164-5056 163-2144 MicroRotofor Cell Lysis Kit (Bacteria), 15 preps, 165-1813 PROTEAN II xi Cell, 20 cm, 1.0 mm spacers (4), includes 50 ml protein solubilization buffer (PSB), 161-0764 Zymogram Sample Buffer, 30 ml 165-8029 Mini-PROTEAN Tetra Cell and Mini Trans-Blot® 15-well combs (2) 25 ml bacteria suspension buffer, 1 ml lysozyme 161-0791 XT Sample Buffer, 4x, 10 ml Module, includes 165-8001 and 170-3935 165-1814 PROTEAN II xi Cell, 20 cm, 0.75 mm spacers (4), (1,500 U/µl) 161-0792 XR Reducing Agent, 1 ml 165-8030 Mini-PROTEAN Tetra Cell for Mini Precast Gels 15-well combs (2) 163-2140 ReadyPrep 2-D Cleanup Kit, 5 preps 161-0719 Tris, 1 kg and Mini Trans-Blot Module, includes 165-8004 ™ 161-0718 Glycine, 1 kg and 170-3935 732-6221 Micro Bio-Spin 6 Columns, includes 25 columns in Tris buffer, 50 collection tubes 161-0301 SDS, 100 g 732-6227 Bio-Spin® 6 Columns, includes 25 columns 161-0416 SDS Solution, 10% (w/v), 250 ml in Tris buffer, 50 collection tubes 166-2404 10% Tween 20, 5 ml 732-6228 Bio-Spin 6 Columns, includes 100 columns 161-0710 2-Mercaptoethanol, 25 ml in Tris buffer, 200 collection tubes 161-0611 Dithiothreitol, 5 g 161-0404 Bromophenol Blue, 10 g 161-0730 Urea, 250 g

86 87 Electrophoresis Guide Appendices

Catalog # Description Catalog # Description Precast Gels Protein Standards Running Buffers and Reagents Recombinant Prestained Protein Standards 161-0732 10x Tris/Glycine/SDS, 1 L 10-Well 15-Well Prep+1 Well 10-Well 12-Well* 9-Well* IPG Well 161-0393 Precision Plus Protein All Blue Standards Description 30 µl 15 µl 450 µl 50 µl 20 µl 30 µl 7 cm IPG Strip 161-0734 10x Tris/Glycine, 1 L Value Pack, 5 x 500 µl ® 161-0744 10x Tris/Tricine/SDS, 1 L Ready Gel Tris-HCl Gels 161-0373 Precision Plus Protein All Blue Standards, 500 µl 5% Resolving Gel 161-1210 161-1211** — 161-1213 161-1214** — — 161-0788 XT MOPS Running Buffer, 20x, 500 ml 7.5% Resolving Gel 161-1100 161-1118 161-1136** 161-1154 161-1172 — — 161-0394 Precision Plus Protein Dual Color Standards 161-0789 XT MES Running Buffer, 20x, 500 ml Value Pack, 5 x 500 µl 10% Resolving Gel 161-1101 161-1119 161-1137 161-1155 161-1173 161-1191** 161-1390** 161-0790 XT Tricine Running Buffer, 20x, 500 ml 12% Resolving Gel 161-1102 161-1120 161-1138 161-1156 161-1174 — 161-1391 161-0374 Precision Plus Protein Dual Color Standards, 161-0793 XT MOPS Buffer Kit, includes 500 ml 20x XT MOPS 15% Resolving Gel 161-1103 161-1121 161-1139** 161-1157 161-1175 — — 500 µl running buffer, 10 ml 4x XT sample buffer, 1 ml 18% Resolving Gel 161-1216 161-1217** — 161-1219 161-1220** — — 161-0397 Precision Plus Protein Dual Xtra Standards 20x XT reducing agent 4–15% Linear Gradient 161-1104 161-1122 161-1140 161-1158 161-1176 161-1194** 161-1392** Value Pack, 5 x 500 µl 4–20% Linear Gradient 161-1105 161-1123 161-1141 161-1159 161-1177 — 161-1393** 161-0796 XT MES Buffer Kit, includes 500 ml 20x XT MOPS 8–16% Linear Gradient 161-1222 161-1223 — 161-1225 161-1226 — 161-1394 161-0377 Precision Plus Protein Dual Xtra Standards, running buffer, 10 ml 4x XT sample buffer, 1 ml 10–20% Linear Gradient 161-1106 161-1124 161-1142** 161-1160 161-1178 — 161-1395** 500 µl 20x XT reducing agent Ready Gel IEF Gels 161-0395 Precision Plus Protein Kaleidoscope Standards 161-0797 XT Tricine Buffer Kit, includes 500 ml 20x XT pH 3–10 161-1111 161-1129** — 161-1165** — — — Value Pack, 5 x 500 µl MOPS running buffer, 10 ml 4x XT sample buffer, pH 5–8 161-1112** — — — — — — 161-0375 Precision Plus Protein Kaleidoscope Standards, 1 ml 20x XT reducing agent Ready Gel Zymogram Gels 500 µl 161-0761 10x IEF Anode Buffer, 250 ml 10% Zymogram Gel with Gelatin 161-1113 161-1131** — 161-1167 161-1185** — — 161-0399 Precision Plus Protein WesternC Standards 161-0762 10x IEF Cathode Buffer, 250 ml 12% Zymogram Gel with Casein 161-1114** — — 161-1168** — — — Value Pack, 5 x 250 µl 161-0765 Zymogram Renaturation Buffer, 125 ml 161-0376 Precision Plus Protein WesternC Standards, 161-0766 Zymogram Development Buffer, 125 ml Description 250 µl 161-0729 EDTA, 500 g 161-0398 Precision Plus Protein WesternC 161-0718 Glycine, 1 kg 10-Gels/Box (Standards + HRP) Value Pack, 5 x 250 µl 161-0713 Tricine, 500 g 10-well 10-well 15-well IPG/Prep 12-well 8+1-well 161-0385 Precision Plus Protein WesternC 161-0719 Tris, 1 kg 30 µl 50 µl 15 µl 7cm IPG Strip/450 µl 20 µl 30 µl

TABLE CONTENTS OF (Standards + HRP), 250 µl Gel Casting Buffers and Reagents Mini-PROTEAN® TGX™ Resolving Gels Recombinant Unstained Protein Standards 161-5100 SDS-PAGE Reagent Starter Kit, includes 7.5% 456-1023 456-1024 456-1026 456-1021 456-1025 456-1029 161-0396 Precision Plus Protein Unstained Standards 100 g acrylamide, 5 g bis, 5 ml TEMED, 10% 456-1033 456-1034 456-1036 456-1031 456-1035 456-1039 Value Pack, 5 x 1000 µl 10 g ammonium persulfate 12% 456-1043 456-1044 456-1046 456-1041 456-1045 456-1049 161-0363 Precision Plus Protein Unstained Standards, 161-0100 Acrylamide, 99.9%, 100 g 4–15% 456-1083 456-1084 456-1086 456-1081 456-1085 456-1089 1000 µl 161-0120 Acrylamide/Bis Powder, 19:1, 30 g 4–20% 456-1093 456-1094 456-1096 456-1091 456-1095 456-1099 Any kD 456-9033 456-9034 456-9036 456-9031 456-9035 456-9039 Natural Prestained SDS-PAGE Protein Standards 161-0122 Acrylamide/Bis Powder, 37.5:1, 30 g 161-0324 Kaleidoscope Prestained Standards, 500 µl 161-0140 40% Acrylamide Solution, 500 ml Mini-PROTEAN® TGX Stain-Free™ Gels 161-0305 Prestained SDS-PAGE Standards, 161-0144 40% Acrylamide/Bis Solution, 19:1, 500 ml 7.5% 456-8023 456-8024 456-8026 456-8021 456-8025 456-8029 low range, 500 µl 161-0146 40% Acrylamide/Bis Solution, 29:1, 500 ml 10% 456-8033 456-8034 456-8036 456-8031 456-8035 456-8039 161-0309 Prestained SDS-PAGE Standards, 161-0148 40% Acrylamide/Bis Solution, 37.5:1, 500 ml 12% 456-8043 456-8044 456-8046 456-8041 456-8045 456-8049 Any kD 456-8123 456-8124 456-8126 456-8121 456-8125 456-8129 high range, 500 µl 161-0154 30% Acrylamide/Bis Solution, 19:1, 500 ml 161-0318 Prestained SDS-PAGE Standards, 161-0156 30% Acrylamide/Bis Solution, 29:1, 500 ml Mini-PROTEAN Tris-Tricine Gels (Pack of 2) broad range, 500 µl 161-0158 30% Acrylamide/Bis Solution, 37.5:1, 500 ml 16.5% Resolving Gel 456-3063 456-3064 — — 456-3065* 456-3066 10–20% Resolving Gel 456-3113 456-3114 — — 456-3115* 456-3116* Natural Unstained SDS-PAGE Protein Standards 161-0200 Bis Crosslinker, 5 g 161-0303 SDS-PAGE Standards, high range, 200 µl 161-0800 TEMED, 5 ml Mini-PROTEAN TBE Gels (Pack of 2) 161-0304 SDS-PAGE Standards, low range, 200 µl 161-0798 Resolving Gel Buffer, 1.5 M tris-HCl, pH 8.8, 1 L 5% TBE Gel 456-5013 456-5014* — — 456-5015* 456-5016 10% TBE Gel 456-5033 456-5034* — — 456-5035 456-5036 161-0317 SDS-PAGE Standards, broad range, 200 µl 161-0700 Ammonium Persulfate (APS), 10 g 15% TBE Gel 456-5053* 456-5054 — — 456-5055* 456-5056 161-0799 Stacking Gel Buffer, 0.5 M tris-HCl, pH 6.8, 1 L 161-0326 Polypeptide SDS-PAGE Standards, 200 µl 4–20% TBE Gel 456-5093* 456-5094* — — 456-5095* 456-5096* Mini-PROTEAN TBE-Urea Gels (Pack of 2) 10% TBE-Urea Gel 456-6033* — — — — 456-6036* 15% TBE-Urea Gel 456-6053* — — — 456-6055* 456-6056* All formats are available as both ten packs (catalog numbers listed) and two packs. To order as a two pack, add an “S” to the end of the catalog number for the corresponding ten pack.

88 89 Electrophoresis Guide Appendices

Catalog # Description Catalog # Description

12+2 Well** 18-Well 26-Well* Prep+2 Well** IPG+1 Well** Gel Casting Accessories Imaging Systems Description 45 µl 30 µl 15 µl 800 µl 11 cm IPG Strip See catalog or www.bio-rad.com for a complete listing of 170-7983 GS-800™ USB Calibrated Densitometer, PC or Mac, 100–240 V Criterion™ TGX™ Gels** accessories, including available empty gel cassettes and glass plates, 7.5% 567-1023 567-1024 567-1025 — — spacers, combs, etc. 170-8195 Gel Doc™ XR+ System with Image Lab Software, 10% 567-1033 567-1034 567-1035 — — 165-5131 AnyGel Stand, 6-row, holds 6 PROTEAN® gels, PC or Mac, includes darkroom, UV transilluminator, 12% 567-1043 567-1044 567-1045 — — 12 Criterion gels, or 18 Ready Gel® mini gels epi-white illumination, camera, cables, Image Lab 18% 567-1073 567-1074 567-1075 567-1072 567-1071 software 165-4131 , holds 1 PROTEAN gel, 4–15% 567-1083 567-1084 567-1085 567-1082 567-1081 AnyGel Stand, single-row 170-8270 Gel Doc EZ System with Image Lab Software, PC 4–20% 567-1093 567-1094 567-1095 567-1092 567-1091 2 Criterion gels, or 3 Ready Gel mini gels or Mac, includes darkroom, camera, cables, Image 8 –16% 567-1103 567-1104 567-1105 567-1102 567-1101 165-4122 Model 485 Gradient Former and Mini-PROTEAN Lab software; samples trays (#170-8271, 170-8272, 10 –20% 567-1113 567-1114 567-1115 567-1112 567-1111 3 Multi-Casting Chamber, includes 165-4120 170-8273, or 170-8274) are sold separately; sample Any kD 567-1123 567-1124 567-1125 567-1122 567-1121 and 165-4110 trays are required to use the system Criterion™ TGX Stain-Free™ 165-4123 Model 495 Gradient Former and PROTEAN 170-8265 ChemiDoc™ XRS+ System with Image Lab Gels** Plus Multi-Casting Chamber, includes 165-4121 Software, PC or Mac, includes darkroom, UV 7.5% 567-8023 567-8024 567-8025 — — and 165-4160 10% 567-8033 567-8034 567-8035 — — transilluminator, epi-white illumination, camera, Total Protein Gel Stains ™ 12% 567-8043 567-8044 567-8045 — — power supply, cables, Image Lab software 161-0786 Bio-Safe™ Coomassie Stain, 1 L 18% 567-8073 567-8074 567-8075 567-8072 567-8071 170-8280 ChemiDoc MP System with Image Lab Software, 4–15% 567-8083 567-8084 567-8085 567-8082 567-8081 161-0787 Bio-Safe Coomassie Stain, 5 L PC or Mac, includes darkroom, UV transilluminaator, 4–20% 567-8093 567-8094 567-8095 567-8092 567-8091 161-0435 Coomassie Brilliant Blue R-250 Staining epi-white illumination, camera, power supply, cables, 8–16% 567-8103 567-8104 567-8105 567-8102 567-8101 Solutions Kit, includes 1 L Coomassie Brilliant Blue ImageLab™ software 10 –20% 567-8113 567-8114 567-8115 567-8112 567-8111 R-250 staining solution, 2 x 1 L Coomassie Brilliant 170-9460 PharosFX™ Plus System, PC or Mac, 110–240 V, Any kD 567-8123 567-8124 567-8125 567-8122 567-8121 Blue R-250 destaining solution includes Quantity One® software, sample tray set, Criterion XT Bis-Tris Gels*** 161-0436 Coomassie Brilliant Blue R-250 Staining Solution, 1 L fluorescence filters (170-7866, 170-7896) and 10% Resolving Gel 345-0111 345-0112 345-0113 — 345-0115 phosphor imaging filters, USB2 cable 161-0438 Coomassie Brilliant Blue R-250 Destaining 12% Resolving Gel 345-0117 345-0118 345-0119 345-0120† 345-0121 Solution, 1 L 170-9450 PharosFX System, PC or Mac, 110–240 V, includes 4–12% Resolving Gel 345-0123 345-0124 345-0125 345-0126† 345-0127 Quantity One software, sample tray set, fluorescence 161-0400 Coomassie Brilliant Blue R-250, 10 g TABLE CONTENTS OF Criterion XT Tris-Acetate Gels filters (170-7866, 170-7896), USB2 cable 7% Resolving Gel 345-0135 345-0136† 345-0137† — — 161-0406 Coomassie Brilliant Blue G-250, 10 g 170-9400 Personal Molecular Imager™ (PMI) System, PC 3–8% Resolving Gel 345-0129 345-0130 345-0131 — 345-0133† 161-0443 Silver Stain Kit, includes oxidizer concentrate, silver or Mac, 110/240 V, includes Quantity One software, Criterion Tris-HCl Gels reagent concentrate, silver stain developer, stains sample tray set, USB2 cable 5% Resolving Gel 345-0001 345-0002 345-0003† — — 20 full size or 48 mini gels Analysis Software 7.5% Resolving Gel 345-0005 345-0006 345-0007 345-0008 — ™ 161-0449 Silver Stain Plus Kit, includes fixative enhancer 170-9690 Image Lab Software 10% Resolving Gel 345-0009 345-0010 345-0011 345-0012† 345-0101 concentrate, silver complex solution, reduction † 170-9600 Quantity One 1-D Analysis Software, PC or Mac 12.5% Resolving Gel 345-0014 345-0015 345-0016 345-0017 345-0102 moderator solution, image development reagent, 15% Resolving Gel 345-0019 345-0020 345-0021 345-0022† — development accelerator reagent, stains 13 full size 170-9630 PDQuest Advanced 2-D Analysis Software 18% Resolving Gel 345-0023 345-0024 345-0025 345-0026† — or 40 mini gels Gel Drying and Electroelution Supplies 4–15% Linear Gradient 345-0027 345-0028 345-0029 345-0030† 345-0103 161-0496 Oriole™ Fluorescent Gel Stain, 1x solution, 1 L 165-1771 GelAir™ Drying System, 115 V, 60 Hz, includes 4–20% Linear Gradient 345-0032 345-0033 345-0034 345-0035 345-0104 165-1777, 2 drying frames, 16 clamps, assembly 8–16% Linear Gradient 345-0037 345-0038 345-0039 345-0040† 345-0105 161-0492 Flamingo™ Fluorescent Gel Stain, 10x solution, table, 50 precut sheets of cellophane support, 10–20% Linear Gradient 345-0042 345-0043 345-0044 345-0045† 345-0107 500 ml gel drying solution 10.5–14% Linear Gradient 345-9949 345-9950 345-9951 — 345-0106 170-3125 SYPRO Ruby Protein Gel Stain, 1x solution, 1 L 165-1777 GelAir Dryer, 115 V, gel drying oven only Criterion Stain-Free Gels 161-0440 Zinc Stain and Destain Kit, includes 125 ml of 10x 165-1251 Whole Gel Eluter with Harvesting Box, includes lid, 10% Tris-HCI Gel 345-1012 345-1018 — — — zinc stain solution A, 125 ml of 10x zinc stain solution electrodes, elution chamber core, base, roller, ruler, 4–20% Tris-HCL Gel 345-0412 345-0418 345-0426 — — B, 125 ml of 10x zinc destain solution 8–16% Tris-HCL Gel 345-8162 — 345-8166 — 345-8161 template, 75 pieces of lower filter paper, 50 pieces 161-0470 Copper Stain and Destain Kit, includes 125 ml of upper filter paper, 50 sealing strips, 25 pieces Criterion Tris-Tricine Gels of 10x copper stain, 125 ml of 10x copper of cellophane, application note; requires a vacuum 16.5% Tris-Tricine 345-0063 345-0064 345-0065† 345-0066† — destain solution source pulling 5" Hg for the harvesting box 10–20% Tris-Tricine 345-0067 345-0068 345-0069 — — High-Throughput Stainers 165-1256 Mini Whole Gel Eluter with Harvesting Box, Criterion IEF Gels ™ 165-3400 Dodeca Stainer, large, 100–240 V, includes includes lid, electrodes, elution chamber core, base, pH 3–10 345-0071† 345-0072† 345-0073† — — 13 trays (12 clear, 1 white), 12 tray attachments, roller, ruler, template, 75 pieces of lower filter paper, pH 5–8 — 345-0076† — — — shaking rack, solution tank, lid with shaker motor, 50 pieces of upper filter paper, 50 sealing tabs, Criterion Zymogram Gels shaker control unit, gel clip 25 pieces of cellophane, application note; requires a 10% Zymogram Gel with Gelatin 345-0079† 345-0080† 345-0081† — — 165-3401 Dodeca Stainer, small, 100–240 V, includes vacuum source pulling 5" Hg for the harvesting box 12.5% Zymogram Gel with Casein 345-0082† 345-0083† 345-0084† — — 13 trays (12 clear, 1 white), 12 Criterion tray 165-2976 Model 422 Electro-Eluter, includes electro-eluter * Multichannel pipet compatible. attachments, shaking rack, solution tank, lid with module, membrane caps (MW cutoff 12,000–15,000), ** Includes reference well(s). shaker motor, shaker control unit, gel clip glass tubes, frits, silicone adaptors, grommets and *** Purchase of this product is accompanied by a limited license under U.S. Patent Numbers 6,143,154; 6,096,182; 6,059,948; 5,578,180; stoppers, buffer tank, lid with power cables 5,922,185; 6,162,338; and 6,783,651 and corresponding foreign patents. † Please allow up to 2 weeks for delivery.

90 91

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124

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061 09 804 22 00 Netherlands Netherlands

7 495 721 14 047 495 14 721 09 Brazil 385 55 11 12700 Switzerland

Finland 44 00 52 10 Finland 52 555 488 The 7670 08 555 08 Russia 7700 472 21 Russia 351

01 877 89 877 Belgium01 01 Mexico 4460

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34 590 91 5200 Sweden Korea

48 22 331 99 99 Portugal 61 2 9914 28002 9914 61 Austria 30 210 953230 210 220 Hong Kong 852 2789 3300 Hungary 36 1 459 India 6100 7000 7000

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27 861 27 246 723 Spain Japan 23 38 41 30 23 Poland 38 41

800 88 22 88 020 Kingdom United 8328 2000 089 884 31 0 Greece 800 6723 424 Australia 216091 86 8500 6169 21 Czech Republic 430420 241 532 Denmark

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LabChip and the LabChip logo are trademarks of Caliper Life Sciences, Inc. Bio-Rad Laboratories, Inc. is licensed by Caliper Bio-Rad Laboratories, Caliper Life Sciences, Inc. trademarks of the LabChip logo are LabChip and licensed under are These products use only. technology for research using the LabChip Inc. to sell products Life Sciences, foreign applications, and related and pending patent 5,436,134; and 5,582,977, 5,863,753; 5,658,751; U.S. Patent Numbers in combination sizing macromolecules, use only in detecting, quantitating, and and development patents, for internal research medical, for providing excludes the use of this product use expressly and development internalresearch where with microfluidics, or clinical analysis, in any event in clinical information services, or providing analysis, or screening diagnostic, or any other testing, 6050

South Africa South 3188 65 6415 963

905 364 3435 China 886 7189 2 2578 01 47 95 47 6901 65 Germany 03

Purchase of Criterion XT Bis-Tris gels, XT MOPS running buffer, XT MES running buffer, XT MOPS buffer kit, and XT MES buffer kit is accompanied kit is accompanied kit, and XT MES buffer buffer XT MOPS buffer, XT MES running buffer, gels, XT MOPS running XT Bis-Tris of Criterion Purchase and 6,783,651, and 5,922,185; 6,162,338; 6,059,948; 5,578,180; 6,143,154; 6,096,182; under U.S. Patent Numbers by a limited license patents. foreign corresponding party. an unrelated return for compensation by CA, for use only by the buyer of the product. Corporation, Carlsbad, Life Technologies sold under license from are standards Plus Protein Precision or its components. this product to sell or resell The buyer is not authorized GmbH to sell Laboratories, Inc. is licensed by Institut für Bioanalytik by German patent application P 19641876.3. Bio-Rad is covered StrepTactin use only. for research these products U.S. Patent Number use only under for research Corporation to sell SYPRO products is licensed by Invitrogen Bio-Rad Laboratories, Inc. 5,616,502. trademarks of and SYPRO are Pro-Q, NuPage, Healthcare. of BASF Aktiengesellschaft. Cy is a trademark of GE Coomassie is a trademark is a Corporation. Tygon is a trademark of Dow Chemical für Bioanalytik GmbH. Triton Corporation. Strep-tag is a trademark of Institut Invitrogen Performance Plastics Corporation. trademark of Saint-Gobain Singapore Taiwan Taiwan 64 9 415 2280 Norway 64 9 415 Zealand New Canada Canada Israel France France Web site USA www.bio-rad.com Bio-Rad Laboratories, Inc.

Bulletin 6040 Rev A US/EG Life Science Group

92 Electrophoresis Guide

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