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A Guide to Polyacrylamide Gel Electrophoresis and Detection

A Guide to Polyacrylamide Gel Electrophoresis and Detection

A Guide to Electrophoresis and Detection

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Table of Contents of Table

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Fluorescent Gel Stain Coomassie Stain Coomassie ™ ™ Fluorescent Gel Stain ™ Pour the Stacking Gel Pour the Resolving Gel ( Disruption) (Cell Lysis Solubilization Preparation PAGE for Cells Cultured Cultured CellsMonolayer

icrofuge Assay Protocol ml) (1.5 eneral Tips for Sample Preparation Sample for eneral Tips eneral Protocols: SDS-PAGE rotein Fractions from from Fractions rotein tandard Assay Protocol (5 ml) ilver (Bio-Rad Stain) G S M G S P Bio-Safe Single-Percentage Single-Percentage Gels Gradient Oriole Flamingo Human Cells Human Tissue Leaves Mammalian Cultures Plant Microbial olecular Weight Estimation olecular Weight el Casting Reagents Casting el andcasting Polyacrylamide Gels uffer Components uffer Components unning Buffers ample Buffers ample Quantitation ( ample ample Preparation Buffersample ample Preparationample erforming Electrophoresis otal Protein Staining Protein otal S R B T S P S G S M H

Glossary References and Related Reading Part Methods II: Protocols Part III: Troubleshooting Preparation Sample Gel Casting and Sample Loading Electrophoresis Staining Protein Total Separation of Evaluation Part IV: Appendices Information Ordering Buffer Formulations

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ulti-Casting Chambers olecular Weight (Size) Estimation (Size) olecular Weight ther Factors Affecting Factors Electrophoresisther uantitation uantitation radient Formers seful Equations seful remade Buffers and Reagents Buffers and remade pecific Protein Stains Protein pecific electing Power Supply Settings aemmli (-HCl) luorescent secondary antibodies for multiplex western blotting western multiplex secondaryluorescent for antibodies oule oule Heating ris-Acetate ris-Acetate ris- otal Protein Stains Protein otal otal Protein Normalization maging Systems maging maging Software mmun-Star AP & HRP Secondary HRP mmun-Star Conjugates & AP Dodeca S G S T M I F U J O PrecisionAb M Q T L Bis-Tris T T IEF I I P AnyGel

Imaging Imaging Analysis Chapter 7 Downstream Applications Blotting (Immunoblotting)Western Immunodetection Electroelution 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 Chapter 6 and Analysis Stains Protein

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Blue Native (BN-PAGE) PAGE Zymogram PAGE ther Types of PAGE iscontinuous Native PAGE Native iscontinuous D O SDS-PAGE mon for Protein Solubilization Solubilization Protein for Solutions mon rmat (Size and Comb Type) oelectric (IEF) Focusing etergents haotropic Agents haotropic uffers and Salts ecombinant Standards ecombinant educing Agentseducing rotein Assays recast vs. Handcast ercentage ower Supplies for PAGE Applications Applications PAGE for Supplies ower lectrophoresis Cells -D Electrophoresis-D R P Is 2 D B Com Polymerization P P Fo Polyacrylamide (PAGE) P E R C

Polyacrylamide Gels Polyacrylamide Chapter 3 Sample Preparation Preparation Sample 3 Chapter Electrophoresisfor General Considerations Cell Disruption Solubilization Protein Removal of Interfering Substances Sample Quantitation (Protein Assays) Selection Reagent 4 Chapter General Considerations Standards Protein Part Theory I: Selection Product and Chapter1 Overview Electrophoresis Works Protein How Workflow General Considerations and Electrophoresis Methods Protein 2 Chapter Instrumentationand Electrophoresis Methods Protein Electrophoresis Cells and Power Supplies Preparation and

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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

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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 Whetherhandcast 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 a concentration 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 . 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 Fig. 1.1. Movement of proteins during electrophoresis. during proteins of Movement 1.1. Fig. Workflow General Considerations and and digestion for mass spectrometric analysis.

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(Figure 1.1). Proteins come in a wide range sizes of (Figure 1.1). and shapes and have charges imparted them to theby dissociation constants their of constituent amino . Asresult, a proteins have characteristic purpose the for exploited be can that rates migration separation.of Protein electrophoresis can be performed in either 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, and (pI), enzymatic activity. In , a significant numbertechniquesof (IEF), electrophoresis, gel 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 themovement to of chargedof in response an electric to field, resulting theirin 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 Product Manual, : A Methods Methods A Proteomics: and Detection, and for Electrophoresis 2-D A Guide Transfer to 2895 bulletin Protein Blotting Guide, Related Literature Related ElectrophoresisGuide

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

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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 analysis Discontinuous Polyacrylamide Gel Electrophoresis, When electrophoresis is performed in 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 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 detergent 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 , 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 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

Other Types of PAGE ® ™ p Bio-Rad’s PROTEAN i12 IEF System provides Vertical electrophoresis cells are made in different size Related Literature Blue Native PAGE (BN-PAGE) individual lane control for up to 12 IPG strips, making formats to accommodate different gels sizes. Deciding BN-PAGE is used to separate and characterize large it possible to run different sample types, different pH which cell to use depends on the requirements for Mini-PROTEAN Tetra Cell protein complexes in their native and active forms. gradients, and multiple protocols at the same time. IEF speed, resolution, and throughput (both the number Brochure, bulletin 5535 Originally described by Schägger and von Jagow can be run under either native or denaturing conditions. of samples and gels) as well as the volume of sample (1987), this technique relies on the solubilization of et hare Native IEF retains and enzymatic available (Table 2.1). Criterion Precast Gel System protein complexes with mild, neutral detergents and Fig. 2.4. Isoelectric focusing. A protein is depicted in a pH activity. However, denaturing IEF is performed in Brochure, bulletin 2710 gradient in an electric field. A pH gradient formed by ampholyte ■■ Mini-format systems — accommodate small gels the binding of negatively charged Coomassie (Brilliant) the presence of high concentrations of , which PROTEAN II xi/XL Cells molecules under the influence of an electric field is indicated. (up to 8.6 x 6.7 cm). The short separation distance Blue G-250 Stain to their surfaces. This imparts a high dissociates proteins into individual subunits and Product Information Sheet, The gradient increases from acidic (pH 3) at the anode to basic maximizes the electrical field strength (V/cm) to yield charge-to-mass ratio that allows the protein complexes (pH 10) at the cathode. The hypothetical protein in the drawing bears abolishes secondary and tertiary structures. Whereas bulletin 1760 rapid separations with moderate resolution. Use these to migrate to the anode as they do in SDS-PAGE. a net charge of +2, 0, or –2, at the three positions in the pH gradient native IEF may be a more convenient option because shown. The electric field drives the protein toward the cathode when systems for rapid analysis, method development, or Coomassie Blue does not, however, denature and it can be performed with a variety of precast gels, it is positively charged and toward the anode when it is negatively when sample volumes are limited. The Mini-PROTEAN® dissociate protein complexes the way SDS does. High- charged, as shown by the arrows. At the pI, the net charge on the denaturing IEF often offers higher resolution and is more System includes the Mini-PROTEAN Tetra Cell (with resolution separation is achieved by electrophoresis protein is zero, so it does not move in the field. The protein loses suitable for the analysis of complex protein . as it moves toward the cathode and becomes progressively into an acrylamide gradient with decreasing pore a capacity of up to four gels) and the high-throughput Related Literature less positively charged. Conversely, the protein gains protons as 2-D Electrophoresis Mini-PROTEAN® 3 Dodeca™ Cell (for running up to sizes; the protein complexes become focused at the it moves toward the anode and becomes less negatively charged. The sequential application of different electrophoresis corresponding pore size limit (Nijtmans et al. 2002, When the protein becomes uncharged (pI), it ceases to move in the 12 gels); both cells are compatible with Mini-PROTEAN field and becomes focused. techniques produces a multi-dimensional separation. 2-D Electrophoresis for Reisinger and Eichacker 2008). Precast Gels Proteomics: A Methods and The most common 2-D technique (O’Farrell 1975) ■■ Midi-format systems — accommodate 13.3 x 8.7 cm Product Manual, Zymogram PAGE Two methods are used to generate a stable, continuous subjects protein samples first to denaturing IEF on gels and offer rapid runs with more samples per bulletin 2651 Zymogram PAGE is used to detect and characterize pH gradient between the anode and cathode: a tube gel or IPG gel strip (for separation by pI), then gel and enhanced separation over mini-format gels. collagenases and other proteases within the gel. ■■ to SDS-PAGE for further separation by molecular Carrier ampholytes — heterogeneous mixtures The Criterion™ System includes the Criterion Cell Gels are cast with or , which acts as a of small (300–1,000 Da) conductive polyamino- weight. High-resolution 2-D methods enable separation ™ TABLE CONTENTS OF (for 1–2 gels) and the high-throughput Criterion substrate for the that are separated in the of thousands of polypeptides in a single slab gel. polycarboxylate compounds that carry multiple Dodeca™ Cell (for 1–12 gels); both cells are gel under nonreducing conditions. The proteins are The resulting spots can be visualized by gel staining, charges with closely spaced pI values. When voltage compatible with Criterion Precast Gels run with denaturing SDS in order to separate them is applied across an ampholyte-containing or they can be transferred to a membrane support ■■ Large-format systems — accommodate large gels by molecular weight. After renaturing the enzymes or gel, the ampholytes align themselves according for total protein staining or analysis with specific (up to 20 x 18.3 cm for the PROTEAN® II System and then allowing them to break down the substrate, to their pIs and buffer the pH in their proximity, antibody detection. For more details, refer to 2-D and 20 x 20.5 cm for the PROTEAN Plus System) zymogram gels are stained with Coomassie (Brilliant) establishing a pH gradient. Ampholytes can be used Electrophoresis for Proteomics (bulletin 2651). Links and offer maximum resolution. The PROTEAN II Blue R-250 Stain, which stains the substrate while in gels (for example, tube gels or vertical gels) or in Electrophoresis Cells and Power Supplies System provides a choice of plates, spacer, leaving clear areas around active proteases. solution (for example, liquid-phase IEF) Electrophoresis Cells and sandwich clamps to cast two gel lengths: Mini Format 1-D Isoelectric Focusing (IEF) Electrophoresis Systems ■■ Immobilized pH gradients (IPG) strips — formed Vertical electrophoresis cells are plastic boxes with 16 or 20 cm. The PROTEAN® Plus Dodeca™ Cell IEF combines the use of an electric field with a pH by covalently grafting buffering groups to a anode and cathode buffer compartments that contain allows maximum throughput with the capability Mini-PROTEAN Precast Gels gradient to separate proteins according to their pI. polyacrylamide gel backbone. A gradient of different electrodes (Figure 2.5). The electrodes (typically to run up to 12 gels at a time It offers the highest resolution of all electrophoresis Mini-PROTEAN Tetra Cell buffering groups generates a stable pH gradient that platinum wire) connect to a jack attached to a techniques (Westermeier 2004). can be tailored for different pH ranges and gradients power supply. The gels are held vertically between Mini-PROTEAN 3 Dodeca Cell When a protein moves through a pH gradient, its net (Bjellquist et al. 1982) the electrode chambers during electrophoresis Midi Format 1-D (Andrews 1986). charge changes in response to the pH it encounters. Electrophoresis Systems Under the influence of an electric field, a protein in a Electrodes pH gradient migrates to a pH where its net charge is Criterion Precast Gels Links zero (the protein’s pI). If the protein moves out of that Criterion Cell position, it acquires a charge and is forced back to the zero-charge position (Figure 2.4). This focusing is Criterion Dodeca Cell Coomassie Stains responsible for the high resolution of IEF. pI values of Lid Large-Format 1-D Coomassie Brilliant proteins usually fall in the range of pH 3–11. Electrophoresis Systems Blue G-250 Stain PROTEAN II xi Cell R-250 Stain PROTEAN II XL Cell

PROTEAN II xi and XL Multi-Cells Running Gel box module PROTEAN Plus Dodeca Cell

Fig. 2.5. Components of a vertical electrophoresis cell. PROTEAN i12 IEF System

12 13 15 Related Literature Related PowerPac Basic 300 V Power 2881 bulletin Flier, Supply High-Current HC PowerPac Power SupplyFlier, 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 and Product Selection Theory Product and PowerPac Basic Power Supply 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- Current Power Supply for mini-format vertical applications PAGE Use the PowerPac HV High-Voltage or PowerPac Universal Power Supply for large-format vertical applications PAGE Use the PowerPac HC Power Supply for applications that require high currents, with such the as PAGE Dodeca Cells high-throughput separated protein is retained within the gel for by example, (for purification further or analysis electroelution). Alternatively,continuous-elution gel electrophoresis using the Model Prep Cell 491 or Mini Prep Cell yields high-resolution separations and proteins in liquid fractions, ready for downstream use. 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 ■■ ■■ ■■ Fig. 2.6. PowerPac Power Supplies.

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C C C C C C C C V or Universal or V Universal or V C or Universal or C Universal or C C or Universal or C niversal asic HC or asic or HC owerPac HC H H H H H H H H H U H B B H H H P Powe Chapter 2: Protein Electrophoresis Protein 2: Chapter and Methods Instrumentation

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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 Preparative ElectrophoresisPreparative ni-PROTEAN Tetra Cell Tetra ni-PROTEAN gh-Throughput Electrophoresis gh-Throughput estern Blotting ini Trans- Cell Trans-Blot ini ini-PROTEAN 3 Dodeca Cell ’Farrell Second Dimension (SDS) Dimension Second ’Farrell riterion Blotter riterion Cell Cell Dodeca riterion pparatus ROTEAN II xi/XL Multi-Cell ROTEAN Plus Dodeca Cell ROTEAN II xi Cell ROTEAN II XL Cell aemmli (SDS), (SDS), aemmli rans-Blot Plus Cell Cell SD rans-Blot rans-Blot Cell rans-Blot C T T T P P W M O Mi C P P Hi M C T A L Wire electrodes electrodes Plate Wire electrodes electrodes Plate transfer High-intensity DNA/RNA Protein

Power Supplies for PAGE Applications PAGE for Supplies Power Power supplies are availablemeet to the power requirements numerous of applications. The choice powerof supply applications for PAGE usually depends on the size and number gels of being 2.2 run. Table compares the Bio-Rad PowerPac Power Supplies applications. vertical electrophoresis recommended for 2.2. PowerPac Table Recommended and echnique

ROTEAN Plus System 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 single gel and the longest range longest the and gel single of separation (with the ability to Offers maximum resolution in a P

ROTEAN II System 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 resolutionover 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 P Trans-Blot Cell Trans-Blot Cell Plus Trans-Blot Cell SD Trans-Blot

riterion System 13.3 x 8.7 x 0.1 cm x 0.1 x 8.7 13.3 Criterion Criterion Dodeca Criterion Gels: Precast Criterion 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 Precast Gels Precast Criterion Criterion Empty Cassettes Empty Criterion Criterion Wire Blotter Wire Criterion Criterion Plate Blotter Cell Trans-Blot Cell Plus Trans-Blot System Turbo Trans-Blot Cell SD Trans-Blot C

System ™ Cell ® Precast Gels Precast Cell Turbo ® ® ® ini-PROTEAN System Mini-PROTEAN Precast Gels: Precast Mini-PROTEAN cm x 0.1 8.6 x 6.7 Gels: Precast Gel Ready cm 8.3 x 0.1 x 6.4 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 Plates Casting Mini-PROTEAN Criterion Blotter Trans-Blot Trans-Blot Trans-Blot SD Cell SD Trans-Blot M

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 ElectrophoresisGuide

TABLE OF CONTENTS 17 Theory and Product Selection Theory Product and Chapter 3: Sample3: Preparation 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

ell • • • • • • — — — C ulture C ammalian M

• • oft — — — — — — — S issues T

• • • — — — — — — lant reen aterial P G M

• — — — — — — — — eeds S

• • • • • — — — — ungi east, east, F Y 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 disruption protocols 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

acteria ■■ ■■ ■■ ■■ ■■ 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 and harsher methods (Table 3.1). B

Chapter 3: Sample3: Preparation ElectrophoresisChapter for

icroorganisms with a mortar and pestle; ethod is usually followed by another sruption method, such as sonication as such method, sruption escription omogenization with either a handheld device (for reaking cells of tissues and pplication of gentle abrasion by vortexing pplication of shear forces by forcing a cell uspension of cells in iso-osmotic solutions uspension of cells in detergent-containing uspension of cells in hypotonic solution; lenders, or other motorized devices; this approach acterial cells); this method is usually followed by isruption of a cell suspension, cooled on ice sually, the mortar is filled with liquid nitrogen and ells with glass beads glass with ells nother disruption method, such as sonication as such method, disruption nother ells, lyticase for yeast cells, and lysozyme for ontaining enzymes that digest the cell wall xample, Dounce and Potter-Elvehjem homogenizers), homogenizers), Potter-Elvehjem and Dounce xample, ells swell and burst, releasing cellular contents reezing in liquid nitrogen and subsequent uspension through a small orifice at high pressure high at orifice small a through uspension olution to solubilize the cell membrane; this membrane; cell the solubilize to olution f ultrasonic waves ultrasonic f he tissue or cells are ground a fine to hawing of cells for example, cellulase and pectinase for plant for pectinase and cellulase example, for s best suited for soft, solid tissues o avoid heating and subjected short to bursts c b i A e m u t H s B A a b c c ( di S s m S t c F D S

o t D

mogenization 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 protease 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 omogenization

■■ ■■ ■■ ■■ ■■ h Glass-bead ho Mechanical Grinding French press

Harsher Methods Sonication

Enzymatic lysis

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

General Considerations Duethe 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)Concentration (as 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 ).

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

18

Sample Preparation Workflow Sample Preparation ElectrophoresisGuide

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

Columns, which 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 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 anysample with a ionictotal strength over 50 mM using columns such as Bio-Spin Micro Common Solutions for Protein Solubilization Solubilization Protein for Solutions Common Ideally, cell lysis and protein solubilization are carried out in the sample bufferthat 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 bufferfinal yield to a 1x buffer concentration. Formulas for various sample buffers are provided in Part II this of guide. Removal of Interfering 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 pHvalues (for example, fractions from most of power separation the diminish chromatography) following the of one Use techniques. electrophoresis methods as needed remove to these contaminants: at a pH suitable for SDS-PAGE. Chapter 3: Sample3: Preparation ElectrophoresisChapter for ME drops and proteins reoxidize, fuzzy 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 or spurious artifactual bands may result. DTT is less volatile and is altered during the reduction reaction form to a ring structure from its protein favors equilibrium The chain. straight original reduction, so lower concentrations DTT of are needed (higher concentrations are recommended for proteins with large numbers disulfideof bonds). tributylphosphine as such Phosphines (TBP) and Tris-carboxyethylphosphine offer (TCEP)* an alternative thiolsto as reducing agents because they can beused 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 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 can be used whenin SDS-PAGE diluted sample with SDS-PAGE buffer. The protein solution should 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

S S S S S S S S Reduction Reduction ME is used in large excess in sample buffers to b ME) and (DTT) disrupt intramolecular and (DTT) intramolecular disrupt ME) dithiothreitol and ME is volatile, evaporates from solution, and reduces b 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-40 and 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 ( 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 , and between free equilibrium an is so 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 insample 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 buffer final yield to buffer a 1x concentration.

If protein phosphorylation be is studied, to include vanadate and fluoride as such inhibitors phosphatase Check the efficacyof cellwall disruptionby light Centrifuge 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 (proteasesare 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 disruptedsamples in solutions containing strong denaturing agents such M 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, usea combination inhibitors of in a cocktail inhibitor protease fluoride (PMSF), aminoethyl-benzene sulfonyl sulfonyl aminoethyl-benzene (PMSF), fluoride ketone chloromethyl lysine tosyl (AEBSF), fluoride (TPCK), etone chloromethyl phenyl tosyl (TLCK), benzamidine, (EDTA), acid ethylenediaminetetraacetic example, (for inhibitors protease 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 where relative amounts protein of be are analyzed, to or in experiments involving downstream immunodetection, the data are only meaningful when the protein compositionis preserved. enzymatic Avoid degradation usingby one or a combination the of following techniques:

20 ElectrophoresisGuide

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

Immunoprecipitation For contaminant removal Bio-Rad offers the following Table 3.2. Bio-Rad protein assay selection guide. SureBeads™ Protein A and Protein G Magnetic (Figure 3.4): Quick Start™ Bradford Bradford DC™ RC DC™ Beads are designed for bioseparation techniques ■■ ReadyPrep™ 2-D Cleanup Kit — uses a modification like immunoprecipitation (IP), co-immunoprecipitation Method of the traditional TCA protein precipitation protocol. Bradford • • — — (co-IP), and protein pull-down assays (Figure 3.3). The kit offers quantitative protein recovery but Lowry — — • • SureBeads Beads are superparamagnetic beads also ensures easy and reproducible removal of Description One-step determination; Standard Bradford Detergent compatible Reducing agent with surface activated hydrophilic and are interfering substances not to be used with high assay, not to be (DC); Lowry assay detergent chemically conjugated to Protein A and Protein G to levels of detergents used with elevated modified to save time compatible (RC DC) ■■ Bio-Spin® and Micro Bio-Spin 6 Columns — provide (>0.025% SDS) levels of detergents specifically bind to the Fc region of immunoglobulin. rapid salt removal in an easy-to-use spin-column (>0.1% SDS) This chemistry enables high IgG binding and low format. Accommodating up to 100 µl of sample, Standard-Concentration Assay nonspecific binding from a variety of biological samples. Sample volume 100 µl 100 µl 100 µl 100 µl these columns remove compounds <6 kD; proteins 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 Product features include: can be eluted in electrophoresis sample buffer Low-Concentration Assay Sample volume 1 ml 800 µl 200 µl 200 µl ■■ Faster IP — using a magnet beads can be collected Sample Quantitation (Protein Assays) Linear range 1.25–25 µg/ml 1.25–25 µg/ml 5–250 µg/ml 5–250 µg/ml faster (within seconds) than with traditional Determine the concentration of protein in a sample assay volume 5 µl 10 µl 5 µl ** centrifugation-based methods (Berkelman 2008) by using protein assays to: Minimum incubation 5 min 5 min 15 min 15 min Assay wavelength 595 nm 595 nm 650–750 nm 650–750 nm ■■ Easier IP — ergonomically designed SureBeads ■■ Ensure that the amount of protein to be separated magnetic rack magnetizes beads in seconds is appropriate for the lane dimensions and visualization method Protein Assays To measure protein concentration in Laemmli buffers, ■■ Use less antibody — unique surface chemistry Related Literature The chemical components of the sample buffer and the use the reducing agent detergent compatible (RC DC™) enables proper antibody orientation for optimal ■■ Facilitate comparison among similar samples; amount of protein available for assay dictate the type of protein assay, which is compatible with reducing binding image-based analysis is simplified when equivalent assay that may be used (Table 3.2). agents and detergents. For more information on protein Modification of Bio-Rad DC ■■ quantities of proteins have been loaded in the lanes Protein Assay for Use with TABLE CONTENTS OF High reproducibility — consistent IgG binding quantitation using colorimetric assays, refer to Bio-Rad of the gel ■■ Bradford Assays (Bradford 1976) — are based on Thiols, bulletin 1909 capacity ensures accurate, reproducible results bulletin 1069. an shift of Coomassie (Brilliant) Blue Colorimetric Proteins Assays, ■■ Low cost to go magnetic — priced similarly to The most commonly used protein assays are G-250 Dye under acid conditions. A redder form of bulletin 1069 colorimetric assays in which the presence of protein leading agarose beads the dye is converted into a bluer form upon binding causes a color change that can be measured with End cap End cap If the sample contains IgG (e.g., tissue lysate, blood- to protein. The increase of absorbance at 595 nm is a spectrophotometer (Sapan et al. 1999, Noble and Reservoir Reservoir derived sample like plasma/serum) that masks the proportional to the amount of bound dye, and thus Bailey 2009). All protein assays utilize a dilution series 3 cm protein of interest during western blotting of the to the amount (concentration) of protein in the 2 cm working bed height ™ of a known protein (usually bovine serum or immunoprecipitated sample, then TidyBlot sample. Compared with other protein assays, 5 cm bovine -) to create a standard curve from 0.8 ml bed volume Secondary Reagent is recommended. g the is less susceptible to 3.7 cm working bed height which the concentration of the sample is derived (for a interference by various chemicals that may be protocol describing protein quantitation, refer to Part II 1.2 ml bed volume Luer end fitting present in protein samples* with snap-off tip of this guide). Porous 30 µm Micro Bio-Spin ■■ Lowry (Lowry et al. 1951) — combines the reactions polyethylene bed Column support retains of cupric ions with peptide bonds under alkaline fine particles 1 4 conditions and the oxidation of aromatic protein Luer end fitting Add SureBeads Magnetize beads, residues. The Lowry method is based on the reaction with snap-off tip Protein A or G remove supernatant, Bio-Spin Column of Cu+, produced by the peptide-mediated reduction ReadyPrep 2-D Links Magnetic Beads. and wash unbound Cleanup Kit protein fractions. of Cu2+, with Folin-Ciocalteu reagent (a mixture of

phosphotungstic acid and phosphomolybdic acid in Sample Buffers 2 5 the Folin-Ciocalteu reaction) and Reagents Add capture antibody, Add elution buffer, magnetize incubate, and magnetize beads, and collect purified ■■ BCA (bicinchoninic acid, Smith et al. 1985) — reacts Protein Assay Kits Links beads to remove target protein. directly with Cu+ (generated by peptide-mediated unbound antibody. and reduction of Cu2+) to produce a purple end product. Fig. 3.4. Bio-Rad products that can be used for contaminant Disposable Cuvettes SureBeads Protein A and The reagent is fairly stable under alkaline conditions removal. Top, Micro Bio-Spin and Bio-Spin Columns; Bottom, for Protein Assays Protein B Magnetic Beads and can be included in the copper solution to make ReadyPrep 2-D Cleanup Kit. 3 the assay a one-step procedure Quick Start Bradford TidyBlot Add sample containing Protein Assay Detection Reagent target protein and incubate. *The Bradford assay is, however, highly sensitive to ionic Bio-Rad Protein Assay Bio-Spin 6 and Micro detergents like SDS. Bio-Spin 6 Columns Fig. 3.3. Immunoprecipitation using SureBeads Magnetic Beads. DC Protein Assay

ReadyPrep 2-D Cleanup Kit RC DC Protein Assay

22 23 25 Theory and Product Selection Theory Product and

Chapter 4: Reagent Selection and4: Preparation Chapter

This chapter details how to select select to how details chapter This (protein reagents the prepare and required buffers) and gels, standards, applications. variousfor PAGE 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 ElectrophoresisGuide

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

MW, kD Select protein standards that offer: Links General Considerations Polyacrylamide Gels Related Literature —250 No particular gel type or buffer is useful for all proteins, ■■ Good resolution of the proteins in the size range Polyacrylamide is stable, chemically inert, electrically and choosing the buffer systems and gel types that of interest —150 neutral, hydrophilic, and transparent for optical Recombinant Protein offer the highest resolution in the size range of interest detection at wavelengths greater than 250 nm. Acrylamide Polymerization — Standards (Markers) ■■ Compatibility with downstream analysis (for —10 0 A Practical Approach, bulletin 1156 may require some experimentation. In selecting These characteristics make polyacrylamide ideal example, blotting) — 75 Precision Plus Protein reagents for PAGE, consider the following: for protein separations because the matrix does not The Little Book of Standards, Unstained Standards Protein standards are available as prestained or bulletin 2414 ■■ Protein standards — select protein standards that — 50 interact with the solutes and has a low affinity for unstained sets of purified or recombinant proteins. In Protein Standards Application Precision Plus Protein provide maximum resolution in the size range of — 37 common protein stains (Garfin 2009). general, prestained standards allow easy and direct Guide, bulletin 2998 Prestained Standards interest and that offer compatibility and utility for Polymerization visualization of their separation during electrophoresis — 25 Increase Western Blot Throughput downstream applications such as western blotting Polyacrylamide gels are prepared by free radical Precision Plus Protein and their subsequent transfer to membranes. Although — 20 with Multiplex Fluorescent Detection, All Blue Standards ■■ Gel percentage — choose the percentage that offers — 15 polymerization of acylamide and a comonomer bulletin 5723 prestained standards can be used for size estimation, the best resolution in the range of interest — 10 cross-linker such as bis-acrylamide. Polymerization Precision Plus Protein Precision Plus Protein unstained protein standards will provide the most — 5 is initiated by (APS) with Dual Xtra Standards—New Protein Dual Color Standards ■■ Handcast vs. precast gels — precast gels offer accurate size determinations. — 2 tetramethylethylenediamine (TEMED) acting as a Standards with an Extended Range greater convenience and superior quality control and Precision Plus Protein Applications and details of Bio-Rad’s protein standards catalyst (Figure 4.2). Riboflavin (or riboflavin-5'- from 2 to 250 kD, bulletin 5956 reproducibility than handcast gels; handcast gels Dual Color Kaleidoscope Dual Xtra All Blue WesternC Unstained Dual Xtra Standards are provided in Table 4.1. ) may also be used as a source of free provide customized percentages and gradients Fig. 4.1. Precision Plus Protein family of protein standards. radicals, often in combination with TEMED and APS. Precision Plus Protein Recombinant Standards Links ■■ Gel format — select mini- or midi-format gels when Polymerization speed depends on various factors Kaleidoscope Standards Recombinant standards are engineered to display throughput is important or sample size is limited; ■■ Precision Plus Protein Unstained Standards — (monomer and catalyst concentration, temperature, specific attributes such as evenly spaced molecular Precision Plus Protein select large-format gels for higher resolution. Select include three high-intensity reference bands and purity of reagents) and must be carefully Coomassie Stains WesternC Standards weights or affinity tags for easy detection. Bio-Rad’s a comb type and gel thickness to accommodate the (25, 50, and 75 kD) and contain a unique affinity controlled because it generates heat and may lead to Coomassie Brilliant Blue recombinant standards are the Precision Plus Protein Precision Protein sample number and volume you are working with Strep-tag, which allows detection and molecular nonuniform pore structures if it is too rapid. R-250 Stain TABLE CONTENTS OF Standards family and are available as stained or StrepTactin-HRP ■■ Buffer system — choose the system that offers the weight determination on western blots. These Coomassie Brilliant Blue Conjugate unstained standards (Figure 4.1). These standards best resolution and compatibility with the protein and standards offer absolute molecular weight accuracy G-250 Stain contain highly purified recombinant proteins with application of interest confirmed by . Because molecular masses of 10–250 kD (or 2–250 kD for the they contain a known amount of protein in each CH Dual Xtra Standards). Protein Standards band, they also allow approximation of protein O NH NH CH H N H N Protein standards are mixtures of well-characterized or concentration. These standards are compatible C C CH C CH C CH recombinant proteins that are loaded alongside protein with Laemmli and neutral pH buffer systems and O O O CH CH CH samples in a gel. They are used to monitor separation are an excellent choice for use with stain-free

as well as estimate the size and concentration of the technology (since they do not contain dye that can N,N’-Methylenebisacrylamide Acrylamide monomer proteins separated in a gel. interfere with stain-free detection). See stain-free cross-linking monomer technology box in Chapter 6 for more details

■■ Precision Plus Protein Prestained Standards Table 4.1. Applications of Bio-Rad’s protein standards. (All Blue, Dual Color, and Kaleidoscope) — include CH CH CH CH CH CH CH CH Precision Plus Protein™ Standards Prestained Natural Standards a proprietary staining technology that provides

™ CO CO CO CO batch-to-batch molecular mass consistency and ™ reproducible migration. The ability to visualize these NH NH NH NH standards makes them ideal for monitoring protein

separation during gel electrophoresis CH Cross-link Dual Color Dual Kaleidoscope Dual Xtra All Blue WesternC Unstained Range High Range Low RangeBroad Natural Kaleidoscope

Electrophoresis ■■ Precision Plus Protein Dual Xtra Standards — NH NH NH NH Accurate MW estimation • • • • • • — — — — prestained standards with additional 2 and 5 kD bands Visualize electrophoresis • • • • • — • • • • CO CO CO CO Orientation • • • — • — — — — — to enable molecular mass estimation below 10 kD Extended MW range — — • — — — — — — — ■■ Precision Plus Protein™ WesternC™ Standards — CH CH CH CH CH CH CH CH Coomassie staining • • • • • • • • • • Fluorescent staining — — — — — • — — — — dual color, prestained, and broad range protein standards that enable detection Polyacrylamide Blotting Monitoring transfer efficiency • • • • • — • • • • when probed with StrepTactin-HRP conjugates; Fig. 4.2. Polymerization of acrylamide monomers and Coomassie staining • • • • • • • • • • the protein standard appears directly on a film or bisacrylamide. Immunodetection — — — — • • — — — — CCD image. Additionally this protein standard has Fluorescent blots* • • • • • — — — — — fluorescent properties that enable detection for MW = molecular weight. * F or use with fluorescent blots, not to be confused with fluorescent total blot stains. Precision Plus Protein fluorescent blots* Prestained Standards contain dyes with fluorescent properties. See bulletin 5723 for details on using precision Plus Protein WesternC Standards for fluorescent multiplexing.

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

Cast the gel Prepare the sample (sample buffer) Fill the electrode reservoirs(running buffer) Chapter 4: Reagent Selection and4: Preparation Chapter ■■ ■■ ■■ Buffer Systems and Gel Chemistries 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: 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,narrow a 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 systems available. systems

0 µl 0 µl 5 µl 0 µl 5 µl 5 µl 00 µl 1 1 2 4 3 3 8 m IPG strip ell Volume 0 µl and 50 µl W 1 cm IPG strip 7 c 3 1

6 0 8 5 2 +1 1 1 1 1 2 2+2 PG I 8 rep+2 1 PG+1 I omb Thickness, mm 1.0 P C

umber of Wells . N

) Gels

for more information. more for ® ® and

® ) ™

Mini-PROTEAN (Criterion 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 placed the 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 be loaded (Table 4.2). The thickness the of gel also plays a role in determining the sample volume that can be loaded. A variety of comb types are available for handcasting; refer to bio-rad.com Table 4.2. Comb types available for Bio-Rad precast polyacrylamide gels. Mini-PROTEAN (Ready Gel (Ready Although handcasting offers the benefit of customized 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 thatthey 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 bio-rad.com

Gels Midi-Format ny ny road ane nstained road Precision Plus Protein nstained Plus Protein Precision MiniPREA G For new or unknown samples, use a broad gradient, for a globalsuch asevaluation 4–20% or 8–16%, theof sample. Then using move to an appropriate single-percentage gel once a particular size range of proteins has been identified ■■ Fig. 4.3. Examples of migration charts. migration Examples of 4.3. Fig. 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 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 less reproducibility with handcast gels.

x 100 x100

Total Total volume, ml g acrylamide + g cross-linkerg + acrylamide g g cross-linker g g acrylamide g + cross-linker Use single-percentage gels separate to bands that are close in molecular weight. Sinceoptimum 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 ■■ ■■ Gels can be made with 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 10–20%. protein migration chartsare 4–15% and tables select to the gel type that offers optimum resolution your of sample (Figure 4.3): 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). 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 referslarger a to %T ratio -to- and smaller average pore sizes. The practical ranges for monomerconcentration are stock solutions 30–40%, of with different ratios of designations The cross-linker. acrylamide to monomer %C = %T =

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

28 ElectrophoresisGuide

TABLE OF CONTENTS 31 Related Literature Related Mini-PROTEAN TGX Precast Sheet, Information Product Gels bulletin 5871 Mini-PROTEAN TGX Precast Gel: Gel A for SDS-PAGE with Improved Stability — Comparison with Standard Laemmli Gels, bulletin 5910 Mini-PROTEAN TGX Precast Gel: Versatile A 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 and4: Preparation Chapter 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 usedwith a Bis-Tris reducing environment and prevent protein reoxidation electrophoresis. during One the of drawbacks usingto 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 donot separate from this front and, therefore, do not resolve discrete into bands. Replacing the in the Laemmli running 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 smallas 1–5 kD can be separated in these gels. 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. Bis-Tris Bis-Tris These systems employ chloride as the leadingion 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 gelswith eitherBis-Tris MES or MOPS denaturing patterns: bufferrunning different migration produces MES buffer is used for small proteins, and MOPS buffer is used for mid-sized proteins. Criterion example, (for Gels Bis-Tris Precast 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

tended X EZ or ™ lycine e lycine G ris- T ( ™ Gels are Laemmli-like, Laemmli-like, Gels are ™ System), andSystem), they 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 Technology in box Chapter 6 for more details). 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 extended shelf life gels that allow rapid fluorescent detection proteins of with the Gel Doc Laemmli (Tris-HCl) (Tris-HCl) Laemmli The Laemmli system has beenthe standard system for SDS- applications and native PAGE for many years. Many researchers use gels 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 movingof 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 andrunning 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

• • • • • — — — — — — — andcast H

.

• • • ­ • • • • • • • • • riterion Gels C

• • • • • • • • — — — — recast (Format) P ni-PROTEAN* Mini-PROTEAN) Mini-PROTEAN) Mini-PROTEAN) Mini-PROTEAN) Mini-PROTEAN) ( ( ( ( ( Mi

unning R T MOPS or T MES T Tricine or ris/glycine/SDS ris/glycine/SDS ris/glycine/SDS ris/Tricine/SDS ris/glycine ris/glycine ris/glycine ris/glycine ris/glycine ris/Tricine/SDS EF cathode and EF anode buffers T

I I T

T T T T T X X X T T T uffers

B

ample ative ative ative ative ative T T or aemmli aemmli aemmli 0% glycerol0% ricine ricine S

L 5

L

N N N T L X X T N N

Ampholytes, allow ®

election Criteria election

S ay be costly olecular weight olecular ffer best separation of high molecular ffer longest shelf life, but reagents ffer best resolution of high molecular ptimized for separating peptide and eight proteins and protein complexes eight proteins; useful in peptide ast with Bio-Lyte etention of native protein structure, asy prepare, to reagents inexpensive enaturing agents, so IEF is performed est choice when long shelf life is needed etection without staining without etection etection without staining staining without etection roteins with molecular weight <1,000 atterns are desired els and need compare to results aemmli-like extended shelf life gels; aemmli-like extended shelf life gels; best aemmli-like gels with trihalo compounds aemmli-like extended shelf life gels with nder native conditions native nder nd readily available; best choice when nd traditional Laemmli traditional separation nd re desired hoice when long shelf life is needed and eparation by protein pl; contain no equencing or mass spectrometry spectrometry mass or equencing witching between precast and handcast esolution of proteins with similar rihalo compounds for rapid rapid for compounds rihalo raditional Laemmli raditional patterns separation rihalo compounds for rapid fluorescence rapid for compounds rihalo or rapid fluorescence detection without detection fluorescence rapid or E a s g s d u L b a p t d O w O p C w s applications R r m L c t a L f staining L L t d O m O

aemmli-like extended shelf life gels with

™

™

F tain-Free H 7.0 GX ris-acetate, ris-acetate,

SDS-PAGE Tris-HCI, pH 8.6 TGX

Stain-Free TGX

T Analysis Peptide Tris-Tricine IE IEF PAGE Native Tris-HCI, pH 8.6 T S TGX Stain-Free T Table 4.3. Gel and buffer chemistries for PAGE. For a current list of precast gels available from Bio-Rad, visit bio-rad.com Type Gel pH 6.4 Bis-Tris, p pH 7.0

30 ElectrophoresisGuide

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

Products for Handcasting Gels Gradient Formers The following products are available to facilitate Gradient gels have a gradient of acrylamide handcasting gels. For detailed handcasting protocols, concentration that increases from top to bottom. To refer to Part II of this guide. create this gradient, the acrylamide solutions must be mixed in a gradient former before being introduced into Premade Buffers and Reagents Related Literature the gel cassette. Typically, two solutions are prepared: Electrophoresis buffers and reagents are available the light solution (equivalent to the lowest %T in the as individual reagents or as premixed gel-casting, range to be poured) and a heavy solution (equivalent Ready-to-Run Buffers and sample, and running buffers. Use of commercially Solutions Brochure, to the maximum %T to be poured). The most common prepared, premixed buffers, which are made with bulletin 2317 gradient gel contains 4–20% acrylamide; however, the electrophoresis-purity reagents and are quality range of acrylamide concentrations should be chosen controlled for reproducible results, saves time but on the basis of the size of the proteins being separated. also maximizes reproducibility, prevents potential mistakes in buffer concentration, and standardizes Two gradient formers are available for PAGE systems. electrophoresis runs. There are no reagents to weigh Depending on the gel format, prepare either a single or filter; just dilute with distilled or deionized water. gel using the gradient former or couple the gradient former with a multi-casting chamber to prepare up AnyGel™ Stands AnyGel Stands (Figure 4.4) provide stabilization and to 12 gels simultaneously (Figure 4.5).

access to gels for casting and sample loading. The ■■ Use the Model 485 gradient former to cast a clamping mechanism secures gel cassettes vertically minimum of 4 mini-format gels at a time using the without excess pressure. They are available in two Mini-PROTEAN 3 Multi-Casting Chamber, or to cast sizes, single- and six-row. a single, large-format (PROTEAN II or PROTEAN

TABLE CONTENTS OF Plus) gel

■■ Use the Model 495 Gradient Former to prepare 4–12 large-format gels (PROTEAN II and PROTEAN Plus) using the multi-gel casting chambers

Fig. 4.4. 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 Polyacrylamide Gel chambers are available for casting gels for the Reagents Mini-PROTEAN, PROTEAN® II, and PROTEAN Premixed Casting Buffers Plus Systems.

Mini-PROTEAN Tetra Fig. 4.5. Multi-casting chambers and gradient formers. Handcast Systems

PROTEAN Plus 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 while resolution optimum provide the of temperature the maintaining during separation. system CHAPTER 5 Performing Electrophoresis

34 ElectrophoresisGuide

TABLE OF CONTENTS 37 Links Power Supplies High-Current HC PowerPac Power Supply PowerPac Universal Power Supply Theory and Product Selection Theory Product and

This yields clear, clear, yields This Chapter 5: Performing Electrophoresis Performing 5: Chapter Increase run 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 ■■ ■■ ■■ General Guidelines for Running Conditions Running for Guidelines General settings different power require cells Electrophoresis with different buffer systems. The valuespresented 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 during long, unsupervisedruns. 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, blot, or 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, dry stained glycerol gels in solution a 10% (storage betweenat 4°C) cellophane sheets. densitometry. for ideal gels publication-quality 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 mayincrease or decrease over the period the of run ■■ ■■ Power Supplies also have an automatic crossover capability that allows the power supply 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 constantduring a run, the voltage, and consequentlypower, the heatthe 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 areused 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 Gel length (increasing gel length demands higher 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 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) ■■ ■■ ■■ ■■ ■■ ■■ ■■ Other Factors AffectingOther Factors Electrophoresis The following variables also changethe resistance theof system and, therefore, affect separation efficiency readings: voltage and current and Joule 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 fromthe system. The amount Joule of heating that occurs depends on the conductivity the of buffer used, the magnitude the of applied field, andtotalthe resistance within the system. The heat generated is proportional the power to (P): consumed 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 using the 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 the of heat that is generated. during extended However, runs or high- conditions,power active buffer cooling is required increases. temperature uncontrolled prevent to /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 thick10–20 V/cm strength of polyacrylamide gels. 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 current: and voltage 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 thetank, filling the tank with running buffer, loading the samples and protein standards, 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 ElectrophoresisGuide

TABLE OF CONTENTS 39 Theory and Product Selection Theory Product and

Chapter 6: ProteinDetection 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 ElectrophoresisGuide

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 Fluorescent stains — fulfill almost all of the In many cases, the choice of staining technique requirements for an ideal protein stain by offering Sensitivity Detection Total Protein Stain (Lower Limit) Time Comments Method depends on the availability of imaging equipment. high sensitivity, a wide linear dynamic range over Rapid Validation of Purified Stain-Free Imaging Fluorescence However, a protein staining technique should offer four orders of magnitude, a simple and robust Proteins Using Criterion Stain Stain-Free 2–28 ng <5 min Rapid; compatible with blotting and mass spectrometry; simple protocol that Uses stain-free compatible Free Gels, bulletin 6001 the following features (Miller et al. 2006): protocol, and compatibility with mass spectrometry. does not require additional reagents; requires tryptophan residues in protein imaging system for detection In comparison to Coomassie or ■■ High sensitivity and reproducibility Coomassie Stains Colorimetric Sensitivity and Protein- techniques, however, fluorescent dyes are more to-Protein Consistency of ■■ Wide linear dynamic range QC Colloidal Coomassie 3 ng 1–20 hr Colloidal endpoint stain; premixed; nonhazardous formulation Flamingo Fluorescent Gel Stain expensive and require either a CCD (charge- Bio-Safe Coomassie G-250 8–28 ng 1–2.5 hr Nonhazardous staining in aqueous solution; premixed, mass spectrometry ■■ Compared to Other Fluorescent Compatibility with downstream technologies coupled device) camera or fluorescence scanner compatible Stains, bulletin 5705 such as protein extraction and assay, blotting, for gel imaging. For these reasons, fluorescent Coomassie Brilliant Blue R-250 36–47 ng 2.5 hr Simple and consistent; mass spectrometry compatible; requires destaining or mass spectrometry with methanol Comparison of SYPRO Ruby stains are often used in proteomics applications and Flamingo Fluorescent ■■ Robust, fast, and uncomplicated protocol and on 2-D gels, where the relative quantitation Silver Stains Colorimetric Gel Stains With Respect of proteins in complex mixtures is performed over Dodeca™ Silver Stain Kit 0.25–0.5 ng 3 hr Simple, robust; mass spectrometry compatible; ideal for use with Dodeca Staining protocols usually involve the following three stainers (Sinha et al. 2001) to Compatibility With Mass several orders of abundance and protein identity is steps (protocols are available in Part II of this guide): Silver Stain Plus™ Kit 0.6–1.2 ng 1.5 hr Simple, robust; mass spectrometry compatible (Gottlieb and Chavko 1987) Spectrometry, bulletin 5754 determined using in-gel proteolytic digestion and Silver Stain (Merril et al. 1981) 0.6–1.2 ng 2 hr Stains glycoproteins, lipoproteins, , nucleic acids Oriole Fluorescent Gel ■■ Protein fixation, usually in acidic methanol or ethanol mass spectrometry. Examples include Flamingo™ Fluorescent Stains Fluorescent Stain: Characterization and (a few staining protocols already contain acid or and Oriole™ Fluorescent Gel Stains Comparison with SYPRO Ruby alcohols for protein fixation and so do not require Oriole Fluorescent Gel Stain* 0.5–1 ng 1.5 hr Rapid protocol, requires no destaining, mass spectrometry compatible; ■■ Silver stains — offer the highest sensitivity, but compatible only with UV excitation Gel Stain, bulletin 5921 this separate step) with a low linear dynamic range (Merril et al. 1981, Flamingo Fluorescent Gel Stain 0.25–0.5 ng 5 hr High sensitivity; broad dynamic range; simple protocol requires no destaining; Bio-Safe Coomassie Stain ■■ Exposure to dye solution Rabilloud et al. 1994). Often, these protocols mass spectrometry compatible; excellent for laser-based scanners Brochure, bulletin 2423 ■■ Washing to remove excess dye (destaining) are time-consuming, complex, and do not offer SYPRO Ruby Protein Gel Stain 1–10 ng 3 hr Fluorescent protein stain; simple, robust protocol;broad dynamic range; mass spectrometry compatible Flamingo Fluorescent Gel Stain sufficient reproducibility for quantitative analysis. Product Information Sheet, Total Protein Stains Negative Stains Colorimetric In addition, their compatibility with mass spectrometry bulletin 5346 Total protein stains allow visualization of the protein Zinc Stain 6–12 ng 15 min High-contrast results; simple, fast, and reversible; compatible with elution or TABLE CONTENTS OF for protein identification purposes is lower than separation pattern in the gel. Table 6.1 compares the blotting as well as mass spectrometry (Fernandez-Patron et al. 1992) that of Coomassie stains and fluorescent dyes advantages and disadvantages of several total Copper Stain 6–12 ng 10 min Single reagent; simple, fast, and reversible; compatible with elution or blotting (Yan et al. 2000) protein staining techniques. as well as mass spectrometry (Lee et al. 1987) ■■ * Do not use Oriole Gel Stain with native gels. Links Negative stains — rapid negative stains require only ■■ Coomassie stains — the most popular anionic ~15 min for high-sensitivity staining, where protein protein dye, Coomassie (Brilliant) Blue stains bands appear as clear areas in a white background. Criterion Precast Gels almost all proteins with good quantitative linearity Zinc and copper stains do not require gel fixation, Stain-Free Technology at medium sensitivity. There are two variants of Criterion TGX Stain-Free ensuring that proteins are not altered or denatured. Bio-Rad’s stain-free technology allows direct Benefits of stain-free technology include: Precast Gels Coomassie (Brilliant) Blue: R-250 (R for reddish), Negative stains can be used as a quality check visualization, analysis, and documentation ■■ Elimination of staining and destaining steps for which offers shorter staining times, and G-250 (G for before transfer to a western blot or analysis by mass Mini-PROTEAN TGX greenish), which is available in more sensitive and of protein samples in PAGE gels without staining, faster results Stain-Free Gels spectrometry, although they are not the best choice destaining, or gel drying. environmentally friendly formulations (Simpson 2010, when quantitative information is desired ■■ Automated gel imaging and analysis Coomassie Stains Neuhoff et al. 1988). Coomassie dyes are also the The system comprises the Gel Doc™ EZ and ■■ Stain-free technology — a haloalkane compound ■■ No background variability within a gel or favorite stains for mass spectrometry and protein ChemiDoc™ MP Imagers, Image Lab™ Software, QC Colloidal in Bio-Rad’s Criterion™, Criterion™ TGX, and Mini- between gels (as is often seen with standard identification. Bio-Rad offers three variations of and precast gels that include unique trihalo Links Coomassie Stain PROTEAN® TGX Stain-Free™ Gels covalently binds Coomassie staining) Coomassie Blue Stains. The QC Colloidal Coomassie compounds that allow rapid fluorescent detection of to tryptophan residues of proteins when activated ■■ Visualization of transferred (blotted) proteins Bio-Safe Coomassie Stain Stain provides sensitivity down to ~3 ng BSA, low proteins — without staining. The trihalo compounds SYPRO Ruby Protein with UV light. This allows protein detection (with a on low-fluorescence PVDF membranes background endpoint staining, and reproducibility react with tryptophan residues in a UV-induced Gel Stain Coomassie Brilliant stain-free compatible imager) in a gel both before that is needed in regulated environments. reaction to produce fluorescence, which can be ■■ Reduced organic waste by eliminating the Blue R-250 Stain and after transfer, as well as total protein detection Dodeca Silver Stain Kit Additionally, this colloidal stain is formulated to be detected by the imager either within gels or on low- use of acetic acid and methanol in staining on a blot when using PVDF membranes (see stain- Coomassie Brilliant ready-to-use (no additional prep or dilution needed) fluorescence PVDF membranes. Activation of the and destaining Silver Stain Plus Kit Blue G-250 Stain free technology box) and to be environmentally friendly (no methanol or trihalo compounds in the gels adds 58 Da moieties ™ Zinc Stain Fluorescent Protein Stains acetic acid waste disposal). Bio-Safe Coomassie Specific Protein Stains to available tryptophan residues and is required for Stain is a nonhazardous formulation of Coomassie Specific protein stains are used to visualize specific protein visualization. Proteins that do not contain Copper Stain Flamingo Fluorescent Blue G-250 that requires only water for rinsing and protein classes such as glycoproteins (Hart et al. 2003) tryptophan residues are not detected. Gel Stain Imaging Systems destaining. It offers a sensitivity that is better than and phosphoproteins (Steinberg et al. 2003, Agrawal The sensitivity of the stain-free system is comparable Oriole Fluorescent Gel Stain conventional Coomassie R-250 formulations and and Thelen 2009), which are of special interest to ChemiDoc MP System equivalent to Coomassie Blue G-250, but with a researchers working in the life sciences (examples to staining with Coomassie (Brilliant) Blue for proteins Gel (left) and blot imaged using stain-free technology. Silver Stains simpler and quicker staining protocol include Pro-Q Diamond and Pro-Q Emerald). with a tryptophan content >1.5%; sensitivity superior Gel Doc EZ System to Coomassie staining is possible for proteins with a Negative Stains Image Lab Software tryptophan content >3%.

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 Flat-bed densitometers — based on high- Dodeca stainers are high-throughput gel staining performance document scanners that have been devices available in two sizes (Figure 6.1): the small size modified to make them suitable for accurate scientific Bio-Rad Imaging Systems accommodates up to 24 Criterion Gels while the large measurement of optical density. Modifications Family Brochure, bulletin 5888 size can accommodate up to 12 large-format gels. The include automatic calibration to traceable reference Imaging Fluorescently stainers ensure high-quality, consistent results and standards, mathematical correction of image non- Stained Gels with Image Lab eliminate gel breakage from excess handling. uniformity, and environmental sealing against liquid Software Quick Start Guide, ™ ™ ™ spills in the laboratory. Densitometers measure the ChemiDoc ChemiDoc ChemiDoc Gel Doc GS-900 Gel Doc bulletin 5989 The stainers feature a shaking rack that holds staining Application MP XRS+ XR+ EZ absorbance (for gels stained with visible dyes) or trays at an angle to allow air bubbles to escape and Blot Detection reflectance (of blots developed with colorimetric ensure uniform gel staining by protecting gels from Multiplex fluorescence ✓ — — — — — ™ breaking. They are compatible with the following stains: reagents) of visible light. Bio-Rad’s GS-900 Chemiluminescence ✓ ✓ ✓ — — — Calibrated Densitometer provides a claibrated linear Stain-free blots ✓ ✓ ✓ ✓ — ✓ ■■ Bio-Safe Coomassie (Brilliant) Blue G-250 Stain dynamic range to a NIST-traceable standard up to Colorimetric ✓ ✓ ✓ — — — ■■ Coomassie (Brilliant) Blue R-250 Stain 3.4 optical density (OD) units SYPRO Ruby Protein Blot Stain* ✓ ✓ ✓ — — — Detection ■■ Flamingo Fluorescent Gel Stain ■■ CCD (charge-coupled device) cameras — operate stain ✓ ✓ ✓ ✓ — ✓ ■■ SYPRO Ruby Protein Gel Stain with either trans-illumination provided by light boxes SYBR® Green I and ✓ ✓ ✓ ✓ ✓ (visible or UV) positioned underneath the gel or blot ® — ■■ Oriole Fluorescent Gel Stain SYBR Safe Stains for imaging a variety of stains (Coomassie, silver, Fast Blast™ DNA Stain ✓ ✓ ✓ ✓ ✓ ✓ ■■ Dodeca Silver Stain Kits Links fluorescence) or epi-illumination detected using Protein Detection, 1-D Gels Imaging colorimetric or fluorescent techniques. Supercooled Stain-free gels ✓ ✓ ✓ ✓ — ✓ CCD cameras reduce image noise, allowing Coomassie blue stain ✓ ✓ ✓ ✓ ✓ ✓ High-Throughput Dodeca Though total protein stains yield visible band Gel Stainers detection of faint luminescent signals. Bio-Rad’s Silver stain ✓ ✓ ✓ ✓ ✓ ✓

TABLE CONTENTS OF patterns, in modern laboratory environments, Gel Doc™ EZ System provides four application- SYPRO Ruby Protein Gel Stain Criterion Precast Gels electrophoresis patterns (called electropherograms) and Flamingo™ and Oriole™ ✓ ✓ ✓ ✓ — ✓ specific trays: a UV tray (for ethidium bromide are digitized by dedicated image acquisition devices Fluorescent Gel Stains staining of DNA gels and fluorescence imaging), a Coomassie Stains and data are analyzed with sophisticated software. Protein Detection, 2-D Gels white tray (for Coomassie, copper, silver, and zinc Coomassie blue stain ✓ ✓ ✓ ✓ ✓ ✓ Bio-Safe Coomassie Stain Once the gels are digitized, the raw data can be stains), a blue tray (for nondestructive nucleic acid Silver stain ✓ ✓ ✓ ✓ ✓ ✓ stored for further reference. Coomassie Brilliant imaging), and a stain-free tray for direct visualization, SYPRO Ruby Protein Gel Stain and Flamingo and Oriole ✓ ✓ ✓ ✓ — ✓ Blue G-250 Stain Imaging Systems analysis, and documentation of protein samples in Fluorescent Gel Stains Selecting image acquisition devices for the digitization polyacrylamide gels without staining, destaining, or Pro-Q Stain ✓ ✓ ✓ ✓ — ✓ Coomassie Brilliant of electrophoresis gels depends on the staining Blue R-250 Stain gel drying (see Stain-Free technology box) Cy2, Cy3, Cy5 Label ✓ — — — — — technique used (see also Table 6.2): ✓ Recommended; — not recommended. Fluorescent Protein Stains * Optimal with low fluorescence PVDF membrane.

Flamingo Fluorescent Imaging Software Gel Stain A robust software package is required for image Gel Doc XR+, Gel Doc EZ, and ChemiDoc XRS+ Oriole Fluorescent Gel Stain acquisition to analyze data and draw conclusions from Imaging Systems. The software allows automatic PAGE applications. Sophisticated gel analysis software configuration of these imaging systems with SYPRO Ruby Protein provides a variety of tools that enhance the user’s appropriate filters and illumination sources. It also Gel Stain ability to evaluate the acquired data. The software allows manual or automated analysis of PAGE gels Silver Stains adjusts contrast and brightness, magnifies, rotates, and western blots resizes, and annotates gel images, which can then be ■■ Quantity One® 1-D Analysis Software — acquires, Dodeca Silver Stain Kit printed using standard and thermal printers. All data in quantitates, and analyzes a variety of data, including Links Imaging Systems the images can be quickly and accurately quantified. radioactive, chemiluminescent, fluorescent, and The software can measure total and average quantities color-stained samples acquired from densitometers, GS-900 Calibrated ChemiDoc MP System Densitometer and determine relative and actual amounts of protein. storage phosphor imagers, fluorescence imagers, Gel imaging software is also capable of determining the and gel documentation systems. The software ChemiDoc XRS+ System Gel Doc EZ System presence/absence and up/down regulation of proteins, allows automatic configuration of these imaging Fig. 6.1. High-throughput Dodeca Gel Stainers. systems with appropriate filters, lasers, LEDs, and Gel Doc XR+ System their molecular weight, pI, and other values. For more information on imagers and gel evaluation software, other illumination sources. It also allows manual or visit bio-rad.com. automated analysis of PAGE gels and western blots ■■ PDQuest™ 2-D Analysis Software — used for 2-D Bio-Rad offers three different software packages for gel gel electrophoretic analysis imaging and analysis:

■■ Image Lab™ Software — image acquisition and analysis software that runs the ChemiDoc MP,

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

■■ Related Literature Analysis The accuracy of MW estimation by SDS-PAGE is in the When possible, separate a dilution series of pure Stain-free technology allows normalization by Beyond protein band patterns, PAGE can yield range of 5–10%. Glyco- and lipoproteins are usually not proteins in parallel. This enables the creation measuring total protein directly in the gel or on the information about a protein’s size (molecular weight) fully coated with SDS and will not behave as expected of a calibration curve (as for molecular weight membrane that is used for western blotting. This Molecular Weight and yield (quantity). Image analysis software greatly in SDS-PAGE, leading to false estimations. For more determination with SDS-PAGE, above) eliminates the need to cut, strip, and reprobe blots Determination by SDS-PAGE, details about molecular weight estimation using required for housekeeping protein normalization bulletin 3133 enhances and facilitates these measurements. ■■ Analyze all samples (including samples for SDS-PAGE, refer to bulletin 3133. strategies and thus saves time and improves the Molecular Weight (Size) Estimation calibration) at least in duplicate Using Precision Plus Protein precision and reliability of western blotting data. Total Standards to Determine SDS-PAGE is a reliable method for estimating the ■■ Use a stain that offers sufficient sensitivity and a high log protein normalization using stain-free technology has Molecular Weight, molecular weight (MW) of an unknown protein, since dynamic range. Fluorescent stains like Flamingo and MW a broader dynamic range (Figure 6.4) and is more bulletin 3144 the migration rate of a protein coated with SDS is Oriole Fluorescent Gel Stains are recommended over effective at detecting small-fold changes in protein Molecular Weight Estimation inversely proportional to the logarithm of its MW. Coomassie and silver staining techniques expression and regulation than normalization using Using Precision Plus Protein The key to accurate MW determination is selecting Linear range Total Protein Normalization housekeeping proteins. WesternC Standards on separation conditions that produce a linear relationship Criterion Tris-HCI and Criterion Western blotting is a widely used method for between log MW and migration within the likely MW Bio-Rad provides imaging systems, software, and gels XT Bis-Tris Gels, bulletin 5763 quantifying protein expression. Changes in expression range of the unknown protein. A protocol for MW levels are identified by comparing band intensities for total protein normalization: Molecular Weight Estimation estimation is provided in Part II of this guide. and Quantitation of Protein between different samples or different experimental ■■ ChemiDoc Imaging Systems – stain-free enabled Samples Using Precision Plus To ensure accurate MW determination: conditions. In order to correct for variations in sample imaging systems available for chemiluminescence Protein WesternC Standards, ■■ preparation, sample loading, and/or transfer efficiency and fluorescence imaging the Immun-Star WesternC Separate the protein sample on the same gel with a Rf researchers need to normalize signal of interest (band) Chemiluminescent Detection set of MW standards (see Chapter 3 for information ■■ Image Lab Software – intuitive software that intensity against a reference. This reference should vary Kit, and the Molecular Imager regarding selection of protein standards) Fig. 6.3. Typical characteristics of a log MW vs. Rf facilitates easy total protein normalization and ChemiDoc XRS Imaging curve for protein standards. only proportionally with the amount of sample loaded. ■■ For statistical significance, generate multiple data protein quantitation using ChemiDoc Imaging System, bulletin 5576 Highly expressed housekeeping proteins, such as , points (>3 lanes per sample) Quantitation Systems TABLE CONTENTS OF ß-tubulin, or GAPDH, are often assumed to be stable Of all the methods available for protein quantitation ■■ ■■ Use a sample buffer containing reducing agents reference proteins and are often used in normalization. Precast and handcast stain-free SDS-PAGE (including UV at 280 nm, colorimetric (DTT or bME) to break disulfide bonds and minimize gels – the unique chemistry of Criterion and the effect of secondary structure on migration dye-based assays, and electrophoresis in combination Mini-PROTEAN TGX Stain-Free gels allows rapid with image acquisition analysis), only protein quantitation fluorescent detection of total protein ■■ Include SDS in the sample buffer. SDS denatures by electrophoresis enables evaluation of purity, yield, secondary, tertiary, and quaternary structures by or percent recovery of individual proteins in complex binding to hydrophobic protein regions. SDS also A Stain-free blot image B ß-actin E Stain-free total protein vs. housekeeping proteins sample mixtures. 50 40 30 20 10 confers a net negative charge on the proteins, which 50 40 30 20 10 6 also results in a constant charge-to-mass ratio Two types of quantitation are possible: relative quantitation Stain-free 5 Actin (quantitation of one protein species relative to the quantity After separation, determine the relative migration GAPDH of another) and absolute quantitation (quantitation of a 4 Tubulin distance (Rf) of the protein standards and of the protein by using a calibration curve generated by a range C ß-tubulin Quantitative Response unknown protein. Rf is defined as the mobility of a 50 40 30 20 10 of known concentrations of that protein). Because proteins 3 protein divided by the mobility of the ion front. Because interact differently with protein stains, the staining the ion front can be difficult to locate, mobilities are intensity of different proteins at identical protein loads 2 normalized to the tracking dye that migrates only can be very different. Thus, only relative quantitative

Links slightly behind the ion front: Relative intensity of protein bands 1 values can be determined in most cases. Absolute protein D GAPDH 50 40 30 20 10 measurements can only be made if the protein under 0 Rf = (distance to band)/(distance to dye front) Image Lab Software investigation is available in pure form and is used as 0 10 20 30 40 50 60 the calibrant. HeLa cell lysate, µg Imaging Systems Using the values obtained for the protein standards, plot

a graph of log MW vs. Rf (Figure 6.3). The plot should For protein quantitation using PAGE to be of value: Gel Doc XR+ System Fig. 6.4. Comparison of protein normalization using stain-free technology and commonly used housekeeping proteins. be linear for most proteins, provided that they are fully ■■ Employ sample preparation procedures that Tenfold dilutions of HeLa cell lysates ranging from 50 to 10 μg were loaded for samples detected with stain-free technology (A) and the Links Gel Doc EZ System denatured and that the gel percentage is appropriate avoid nonspecific protein loss due to insolubility, housekeeping genes β-actin (B), β-tubulin (C), and GAPDH (D). The protein quantification signal is higher with stain-free technology than with housekeeping genes (E). for the MW range of the sample. The standard curve is precipitation, and to surfaces ChemiDoc MP System sigmoid at extreme MW values because at high MW, Fluorescent Protein Stains ■■ Ensure all proteins enter the electrophoretic the sieving affect of the matrix is so large that molecules ChemiDoc XRS+ System separation medium Flamingo Fluorescent Gel Stain are unable to penetrate the gel. At low MW, the sieving Quantity One 1-D effect is negligible and proteins migrate almost freely. ■■ Optimize the quality of the electrophoretic separation. Oriole Fluorescent Gel Stain Analysis Software To determine the MW of the unknown protein band, For example, wavy, distorted protein bands and comigration of bands lead to questionable results Coomassie Stains PDQuest 2-D interpolate the value from this graph. Analysis Software Silver Stains

44 45 47 Theory and Product Selection Theory Product and Chapter 7: DownstreamApplications 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 ElectrophoresisGuide

TABLE OF CONTENTS 49 Links Anti-Housekeeping hFAB Antibodies 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 Cell (or ® II or Mini-PROTEAN Cells) 3 ® Chapter 7: DownstreamApplications 7: Chapter older Mini-PROTEAN older to eluteto from single or multiple gel slices. The 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 through migrate to macromolecules permits but macromolecules the When applied. is current when have passed through thefrit, they are collected in the membrane cap for further analysis or testing. Depending on the buffer system, the Model 422 Electro-Eluter can be used for elution or dialysis up of sixto samples. 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 and a buffer into solution. Once eluted, proteins can be assayed for activity, applied to subsequent purification steps, or subjectedto mass spectrometry or a variety other of applications. The Model Electro-Eluter combines 422 (Figure 7.3) with the tank and lid the of Mini Trans-Blot Fig. 7.3. Model 422 Electro-Eluter 422 Model 7.3. Fig.

) technology. technology. ) ® MP Imaging System, ™ permitting detection multiple of proteins in a single blot. This can save time, sample, and reagents. Fluorescent secondary antibodies for multiplex multiplex secondary for Fluorescent antibodies blottingwestern Secondary Anti-MouseStarBright 700 (Goat Blue Antibodies Easy Sensitivity and Unmatched — Anti-Rabbit) Goat and Multiplexing StarBright Blue (Ex/Em 700 nm/700 is nm) a new = 470 ultra-sensitive fluorescent label that allows detection lowof abundance proteins in seconds exposure of cross-adsorbed Highly background. minimal with time secondary antibodies conjugated StarBright to are ideal for fluorescentwestern blotting — eitherfor the detection a single of target protein or for multiplex detection several of proteins on one blot, without Fluorophore StarBright The reprobing. and stripping is composed a condensed of polymer made up of -emitting and monomers, light-absorbing multiple signal bright exceptionally an provides which fluorophores. traditional most to compared StarBright Blue Fluorescent 700 Secondary Antibodies can be used with traditionalfluorophores like RGB fluorophores and IR 800 dyesfor multiplexing. In addition, StarBright Antibodies can be used with hFAB and/or technology stain-free Bio-Rad’s Primary for Antibodies Anti-Housekeeping Rhodamine protein normalization. These antibodies are optimized ChemiDoc the with use for (Anti-Actin, Anti-Tubulin, Anti-Housekeeping antibodies hFAB worry cross-reactivity about Anti-GAPDH) never and — human are Antibodies Protein Anti-Housekeeping hFAB Fab fragments directly labeled withrhodamine (Ex/ These nm). Em = 530 antibodies nm/570 allow easy, one-step detection common of housekeeping proteins like actin, tubulin, and GAPDH in human, mouse, and rat samples without the need for a secondary antibody. Human Bio-Rad’s using created are These antibodies Library Antibody (HuCAL Combinatorial This ensures no species cross-reactivity, which means they can be used in multiplex western blots with primary antibodies from any host species. Wash Wash Rhodamine* B700*

™

™ based on based conjugated conjugated Transfer Incubate with Incubate Block unbound Block or fluorescenceor AP & HRP Secondary HRP Conjugates & Antibody AP membrane sites membrane primary antibody ™ Develop signal Develop Incubate with Incubate color chemiluminescencecolor secondary ligand or antibody * Fluorescent labeled antibodies exclusive to Bio-Rad Laboratories. Bio-Rad to exclusive antibodies labeled Fluorescent * Fig. Triplex 7.2. western blot imaged by the ChemiDoc MP System. Imaging RedTarget protein — StarBright (ATG7): #1 Target protein Green #2 (AKR1C2): — DyLight 800 Normalization protein (tubulin): Blue — hFAB Immun-Star Bio-Rad’s Immun-Star range offers a suite affinity- of specificity), (high cross-adsorbed purity), (high purified blotting-grade HRP- and AP-conjugated goat anti- secondary for antibodies anti-rabbit goat and mouse easy and sensitive colorimetric or chemiluminescent western blot detection. High titer the of blotting-grade High sensitivity. assay increases conjugates antibody titer also allows greater working dilutions, decreasing ratio. signal-to-noise the increasing and background An ensemble related of product offerings includes AP substrate, substrate packs, and complete detection kits. Fig. 7.1. Western blottingFig. 7.1. workflow. Validated Antibodies for Western Blotting Western for Antibodies Validated ™ occupied protein-binding sites on the membrane membrane the on sites protein-binding occupied econdary antibodies, specificfor the primary roteins are transferred from the gel a membrane to he blot is probed for the proteins interest of with abeled protein bands are visualized the by bound S L P Un T specific primary antibodies. antibody type and conjugated detectable to reporter groups such as enzymes or radioactive isotopes, are used label to the primary antibodies. reporter groups acting on an added substrate or by . where they become immobilized as replica a the of band patterngel’s (blotting).  are saturated prevent to nonspecific bindingof (blocking). antibodies  

Immunodetection PrecisionAb The PrecisionAb Antibody portfolio is a premium primary sensitive and specific highly of collection antibodies that have been extensively validated for western blotting for consistent performance with minimal need for optimization. All antibodies are tested using whole cell or tissue lysates expressing endogenous levels the of target proteins (no overexpression by or target enrichment). A detailed protocol and complete western blot image is provided so that the data can easily be replicated with complete confidence.Trial sizesof antibodies with positive control lysates allow easy access for testing performance before buying larger quantities. Bulk quantities these of antibodies can be ordered by contacting the antibody specialists. 4. 5. 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 information. more for 2895) bulletin (Bio-Rad 2. 3. Western Blotting (Immunoblotting)Western When specific antibodies available,are transferring the proteins a membrane to (blotting) followed by immunological staining is an attractivecomplement generalto protein stains and provides additional experiment typical A immunoblotting information. Following PAGE: consists7.1). five of steps (Figure 1.

Antibodies StarBright Secondary Conjugates Immun-Star HRP and AP PrecisionAb Antibodies Mini-PROTEAN Cell Mini-PROTEAN Mini Trans-Blot Cell Model 422 Electro-Eluter 422 Model Links 48 bulletin 2032 bulletin Reagents Brochure, Brochure, Reagents Western Blotting Detection bulletin 2895 bulletin to Transferto and Detection, Protein Blotting Guide, A Guide Related Literature Related ElectrophoresisGuide

TABLE OF CONTENTS 51 Methods

Part II Methods

50 ElectrophoresisGuide

TABLE OF CONTENTS 53

Methods cells; store and 7 uffer (2x) aline (PBS) use buffer RIPA at 4°C s buffer solubilization RIPA (use 1 ml buffer RIPA with 3 × 10 SDS-PAGE sample b Centifuge Sonicator Phosphate buffered Phosphate Protein Assay Protein ■■ ■■ ■■ ■■ ■■

Equipment Reagents Links DC Assay RC DC Protein Buffer Sample SDS-PAGE ReadyPrep 2-D Cleanup Kit

se a cell scraper collect to 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. U the lysate and transfer a to tube. microcentrifuge 1 2 3 Monolayer Cultured CellsMonolayer entrifuge × g for cell debris at ~14,000 Place the cell suspension on ice, incubate 5 min, and sonicate at lysis Check intervals. appropriate efficacyby light microscopy.  C 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 Pellet the cells centrifugation 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 This protocol uses sonication and radioimmunoprecipitation assay buffer, for cell (RIPA) lysis and protein extraction. Cells Cultured Suspension

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% (DTT) dithiothreitol 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 stated like 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 mixwith “Dilute 3 parts, 1:4,” making 4 parts, of a total diluting the part a quarter 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 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 protein quantitation assay determine to the amount of proteintotal ineach 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

) d a chemical protease inhibitor the lysis to buffer. isrupt the sample or place freshly disrupted samples erform cell disruption at low temperatures diminish to yse samples using at pH >9 either sodium carbonate f protein phosphorylation is to be studied, include include studied, be to phosphorylation is protein f I vanadate and fluoride as such inhibitors phosphatase or Tris as a bufferingor Tris agent in the lysis solution (proteases are often least active at basic pH) Ad fluoride sulfonyl aminoethyl-benzene (PMSF), (for example, leupeptin, pepstatin, aprotinin, and bestatin). For best results, use a combination cocktail inhibitor protease a in inhibitors of in solutions containing strong denaturing agents 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 P enzymatic activity L fluoride phenylmethylsulfonyl Examples include D ketone chloromethyl lysine tosyl (AEBSF), (TPCK), tosylphenylchloromethyletone (TLCK), ethylenediaminetetraacetic acid (EDTA), inhibitors protease peptide and benzamidine, – – – – – – – – – – 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 at least 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 concentration of 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 ElectrophoresisGuide

TABLE OF CONTENTS

55

Methods R 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 cellsis 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 ■■ ■■ ■■ ■■ ■■ and lysis eproducible 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 2-D ™ or Micro ® Columns, which are filled with size ™ Buffer exchange — use Bio-Spin use — Buffer exchange Precipitation — use the ReadyPrep exclusion mediaequilibrated 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 as high 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 ool 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 5,000 x g and resuspend the pellet in an equal PBS and volume 37°C of centrifuge again. Repeattwo 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 C Centrifuge x 10 cells (~5 5 6 1 2 3 4 Protocols Sample Preparation Microbial Cultures This protocol relies on cell lysis with ultrasonic waves in combination with a solubilization in SDS under elevated temperature. This ensures deactivation and proteases. of denaturation

lace 3–4 leaves inthe mortar, add 1 hr at –20°C. 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 room to temperature and perform the protein assay. liquid nitrogen, and grindthe leaves in the liquid nitrogen a fine to powder. leafTransfer powder ml 20 protein of into precipitation solution and incubate for Cool protein precipitation and wash solutions Chill –20°C. to a mortar with nitrogen. liquid P 1 8 9 4 5 6 7 2 3 Leaves Plant minimize the deleteriousTo effects endogenous of which protocol, below the use compounds, plant involves grinding the tissue in a mortar and pestle with liquid nitrogen. entrifuge at 35,000x g for 30 min erform a protein assay ncubate the sample at room mmediately after grinding, transfer C at room temperature. P 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 notheat the sample. I 60 mg tissue powder a to containing tube microcentrifuge mllysis buffer. 1.0 Optional: sonicate the sample on ice 5 times, for 2 sec every time. Pause between sonication steps to avoid overheating. I temperature for 30 min. Vortex from time time. to Chill a mortar with liquid nitrogen, then grind small tissue pieces in the presence liquid of nitrogen a to fine powder. 7 5 6 2 3 4 1 Mammalian Tissue This protocol involves freezing mammalian tissue nitrogen liquid in samples) biopsy example, samples (for at –196°C.

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

W 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 U mortarand 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) ■■ ■■ ■■ ■■ ■■ ■■ precipitate leaves, plant ith se liquid nitrogen and a Links Reagents

Tips 54 SDS-PAGE Sample Buffer Equipment n n Sample Preparation Protocols ElectrophoresisGuide Mammalian Tissue Mammalian Plant Leaves Plant

TABLE OF CONTENTS 57 Methods Product Links: Product U acrylamideespecially monomers, problems polymerization avoid to P casting solution is critical for both polymerization the of reproducibility (oxygen removal) and the avoidance of spectrometry mass to related problems contamination) (keratin A t polymerization; and degassing for equilibrate the stock solutions to room temperature A proceed for at least 2 hr ensure to size pore of maximum reproducibility M performance best for R months becauseit is subject to the causes which oxidation, gradual loss of catalytic activity T free of chips. Clean glass plates with ethanol and lint-free cloths before use T should be at least 2x the height of the sample in the well. This ensures band sharpness, even for diluted samples protein S Do not freeze. Wrap handcast gels tightly in plastic wrap with combs insertedstill R 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 F polymerization, referAcrylamide to Polymerization – A Practical Approach, bulletin 1156 A neurotoxins when in solution. Avoid direct contactwith the solutions and clean up spills F PROTEAN 3 Multi-Casting Chamber PROTEAN II (catalog #1654110), Multi-Gel Casting Chamber (catalog Multi- Plus PROTEAN or #1652025), Casting Chamber (catalog #1654160) se only high-quality reagents, roper degassing and filtering of the emperature of 23–25°C is best should reactions PS/TEMED-initiated ake fresh APS solution every day eplace TEMED every three he glass plates must be clean and he height of the stacking gel tore gels flat in the fridge 4°C. at un handcast gels with discontinuous acrylamide about information more or crylamide bisacrylamide and are or casting multiple gels, use the Mini- n n  n n  n  n  n  n  n  n  n General Tips for Handcasting for Tips General n n — 5 µl .5 µl 5 ml 7 50 µl % 7 1 .75 ml .75 1 X 3 .33 x X ml 6.0 ml 0 cm 8.0 ml 2.0 ml 2.0 4.0 ml4.0 6.0 ml 0 2 1 2 3 4 9 II xiCell 1.03–(0.33 x X) ml 1 ®

— 5 µl .5 µl 5 ml 12% 7 50 µl .0 ml.0 7 1 solving Gel solving .75 ml .75 1 8.4 ml8.4 6 .03 ml 5.6 ml5.6 6.8 ml 9.2 ml 2.8 ml2.8 3 PROTEAN 16 cm 16 1 1 2 3 7 5

Re — ™ 5 µl

.5% .5 µl 5 ml 7 50 µl 7 7 1 .75 ml .75 .75 ml .75 1 .28 ml 7 3 3

ell 5.0 ml5.0 — — 1 — — C Criterion

*

®

l

— % 5 µl 5 µl 9 m 5 ml 1 4 7 50 µl 1 1 .78 ml .78 .98 ml 3 1

tacking Geltacking

S ell

.4 ml.4 .6 ml.6 .2 ml Mini-PROTEAN — 4 5 8 — C

O 2 0 ml of monomer solution is sufficient for two stacking gels of any thickness. 1  .5M Tris-HCl, pH 8.8 Prepare the resolving and stacking gel solutions without APS or TEMED. (Tables consult 1 and 2; the instruction manualfor 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 1.5 mm 3.0 * (GelThickness) 1.0 mm 1.0 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 Table 2. Recipes for stacking and resolving gels. Adjust amounts as needed for the format you are using (see 1). Table SDS 10% diH 1 10% APS 10% 30% Acrylamide/bis 0.5M Tris-HCl, pH 6.8 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 S to 5 μl Reagent A needed. This solution repare 3–5 dilutions a protein of ipet µl protein of standard 25 or 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 each tube and vortex. Incubate the tubes formin 1 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 P mg/ml protein). standard (0.2–1.5 P microcentrifuge clean into sample µl RC of tubes. Add 125 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 lot absorbance measurements as a function of Reagent each S to nterpolate the concentration the of protein samples from Reagent each B to tube P concentration for the standards. I measurements. absorbance sample and plot the Read absorbance each of sample nm. The at 750 are stable for at least 1 hr. Reagent A needed. This Reagent each I into tube and 8 9 10 d 510 µl Reagent of A d 510 d 500 µl RC of d 4 ml DC of ProteinRC DC Assay) repare 3–5 dilutions a protein of Ad and vortex immediately. Incubate at room temperature min. for 15 and vortex. Incubate tubes at room temperature for 5 min, or until the Vortex. dissolved. is precipitate  Add μl DC 20 of 1 ml DC of solution is referred as Reagent to A 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. Ad P 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 roomtemperature. Ad Each standard or sample assayed μl Reagentrequires A 510 7 5 6 2 3 4 1 Standard ml) (5 Assay Protocol

F the standards in the same sample the buffer as P each time the assay is performed

or best results, prepare

repare astandard curve 56 RC DC Protein Assay PROTEAN II xi Cell Mini-PROTEAN Cell Mini-PROTEAN Criterion Cell Criterion Links n n to a standardto 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 and detergent compatible Protein quantitation (DC). can be performedin 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 (Lowrycompatible1951), et (RC) al. Sample Quantitation ( Sample Quantitation Protocols ElectrophoresisGuide

TABLE OF CONTENTS 59 Methods I 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 f gravity flow isn’t fast 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 theon multi-casting chamber. Combine all reagents except the initiators, and degas the solution min. for 15 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 chamber, open 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 lineargradient, 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 6.7 ml 6.7 4 ml .5 ml 75 µl 75 µl 7.5 µl 75 µl 7.5 3.8 ml 3.8 ml .3 ml olume olume 5–90 ml V 05–110 ml 05–110 45–150 ml45–150 8 o Prepare X = 7 X = 1 X = 3 X = 2 X = 2 X = 3 X = 1 X = 4 X = 2 X = 2 t 1 1

0 ml 00 ml 40 ml 8 1 1 or 12 Gelsor 12 f olume Required Required olume V

1.0 mm 1.0 mm 1.5 Plates mm 0.75 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 TEMED APS volume;10% = X µl)/10 (275 Determine the volume acrylamide of prepareto ml 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 Then, manual. instruction Chamber flowwater through the stopcock and the fill to required volume the measure chamber Disassemble the 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% Water ml) ml + 13.8 = X (55 ml) – (7.3 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) (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) 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

d the APS and TEMED the to emove the comb pulling by it R straight up slowly and Rinse gently. the wells completely with diH Dry the area above the separating gel withfilter 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 Ad 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 the to overlay An completely. cassette solution is not necessary for comb a when polymerization is in place. Allow the gel polymerize to 30–45 min. 4 5 6 1 2 3 Pour the Stacking Gel

-butanol. Stacking gel Stacking gelResolving (contd.) O. 2 Add the APSand 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 casting gel 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

D remain onthe 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 alcohol 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 water, n F W gel solution, pour the solution down the middle of the outside plate of the gel sandwich or down theside of one of the spacers. Pour smoothly prevent to it from mixing with air o not allow alcohols to or the overlay solution,

resolving the pouring hen 58 Reagents Polyacrylamide Gel Polyacrylamide Gel Links Tips Handcasting Polyacrylamide Gels Polyacrylamide Handcasting Protocols ElectrophoresisGuide n n n

TABLE OF CONTENTS 61 Methods Links Prot/Elec Tips

Tips fit ™ Coomassie Coomassie ™ The total protein amount loaded perlane depends on the sample complexity and sensitivity of the staining technique. Using 15–20 µg protein per lanefor mini- or midi-format gels is a good starting point for complex protein samples when staining with Bio-Safe >12,000 x g at x 20°C before loading>12,000 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 best results. 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 altersthis balance and leads undesirable to results buffers running reuse not Do V perUse 5–10 cm of gel for about min during 10 sample entry (or until the sample has concentrated at the starting point of the separation gel). Then continue with the voltage setting recommended in the 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 supply that 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

l l — 5 µ 5 µ 0 µl 1 Nonreducing inal 20–31 mA

nitial 35–50 mA I F

l

0 µl 5 µ 1 .75 µl .75 .25 µl ducing

4 0 Re

O. 2

emove the comb and tape from the gels and assemble the unning buffer (1x): Dilute 100 ml of 10x stockunning Dilute ml 10x of 100 buffer (1x): ample buffer: Use Laemmli sample buffer. ill 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 runs buffer.buffer At V, >200 chamberto the 4-gel (800 ml) mark. S R cell. electrophoresis F

R with 900 ml diH

fter electrophoresis is complete, turn the power supply off and aemmli sample buffer -mercaptoethanol otal volume Gel in the Mini-PROTEAN Cell. For detailed Tetra instructions, L T b b. Prepare gels and assemble the electrophoresis cell: a. Prepare samples as indicated in the table below. b. A 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 at 300 decrease V to the run time. 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 protocols on the examples. as pages following Prepare buffers: a. Bio-Rad’s Mini-PROTEAN Gels, TGX the gels can be run ™

TGX ®

7 3 4 5 1 2 6

-Mercaptoethanol 60 Laemmli Sample Buffer Sample Laemmli b PowerPac Basic Power Supply 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 ElectrophoresisGuide 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 Image Lab 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 .0 1 in 5 min 5 min 5 min 0 min 0 min 0 min 0 min 0 min 0 min 2 m 30 min 30 min 1 1 1 1 3 6 ~ ~ ~ 1 1 1.0 mm Gel1.0

> Standards Unknown .8 0

.6 0

f in in in in in R 5 min 5 min 5 min 0 min 0 min 5 min 5 min 5 m 5 m 5 m 5 m 1 m

uration 1 1 2 3 ~ ~ ~ .4 0 .5–1.0 mm Gel 0 own precipitate appears. precipitate own br

.2 0

= 0.997 30 sec. Develop until solution turns yellow or until 1 2 3 4 5 6 7 8 2 D

in in in in 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 — —

5 min 5 min 0 min 5 min 5 min 5 min ™ 3 m 2 m 2 m 2 m 1 1 1 3 ~ ~ 0 15 10 75 37 25 20 50 0 0.5 mm Gel

150 100 250

1.0 2.0 3.0 < 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 shownis the actual size. unknown protein (GFP). Proteins were separated by SDS-PAGE in a Criterion

~

graphically 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml 00 ml f olume 2 2 2 2 2 4 4 4 4 4 4 4 4 V

. f

value using the the using value f

O O 2 2

top

iH iH Software (or equivalent). Software (or xidizer S ixative ixative eagent d d eveloper F F ™ O R D ilver reagent = migration distance the of protein/ % acetic acid f S 5 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 bandand the to front. dye For each band in the standards, R the calculate 0% ethanol/5%0% acetic acid 1 0% methanol/10%0% acetic acid ellow color is gone from the gel) 4 y Repeat washes 5–7 times until all the ( 1 2 3 4 5

0 3 2 1 3 4 5 2 1 8 9 6 7 1 1 1 1

Step 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

or using Image Lab Silver StainingSilver (Bio-Rad Stain) Silver

O prior to imaging. to prior O 2 O). Cover the tray, place Cover on the a rocker tray, O). 2 our off the fix solution and add 50 ofml 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 and agitate gently min. for 10 Rinse gel with diH Place gel in a staining ml tray with of 100 fixing solutionacetic10% ethanol,(40% place acid). Coveron the a rocker, tray, and agitate gently for at least 2 hr. P 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 emove all water fromstaining 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 R 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 gel diHTransfer to 400 ml methanol of the L bottle 1 to diluents.of Then ml Oriole of add10 Fluorescent Gel Stain concentrate and If using the 5 L configuration, prepare Oriole Stain solution adding by Wash gels three times for 5 min each in 200 ml diH imaging. Destaining is not necessary. not Destaining is imaging. Stained gels can be stored in water. Coomassie Stain ™ FluorescentGel Stain ™ 3 2 3 1 1 2 Total Protein Staining Protein Total General protocols are described below Gels. Mini-PROTEAN for For more details, referthe instruction to manual for thestain you are using. Bio-Safe Protocols Oriole O 2

F shrink-wrap the stained gels glycerolin a 10% solution 4°C) at (storage Flamingo and Oriole Oriole and Flamingo Fluorescent Gel Stains have a higher dynamic range than Coomassie or silver staining techniques, making them quantitative for suitable analysis protein highly purified diH purified highly F U G on a horizontal shaker, making sure the gel is completely covered with stain solution all the time U containers for gel staining. If possible, place a lid on the container avoid to contamination of the solution staining A the staining process. to Try avoid touching the gels with gloves Wet fingers. your with water or bufferbefore handling the gel keep to the gel from sticking and tearing dyes can be counterstained counterstained be can dyes Coomassie colloidal with furtherfor reference. In enhances so doing fact, colloidal the of sensitivity Stain Coomassie G (conductivity <2 μS) <2 (conductivity

els stained with fluorescent or long term-storage, luorescent dyes like se pure chemicals and ently agitate the container se clean and dust-free

lways wear gloves during 6262 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 

ElectrophoresisGuide ElectrophoresisGuide     TIPS  

TABLE OF CONTENTS 65 Methods

.0 ml .0 .0 ml .0 ml .0 .3 ml ml .3 .3 ml ml .3 .75 ml .75 .75 ml .75 2.0 ml 2.0 4.0 ml 4.0 5.0 ml 5.0 6 6 6 0 0 2.0 mg 2.0 1 3 3 1 2 o 30 ml o 30 ml o 30 ml 1 t t t -mercaptoethanol -mercaptoethanol

-mercaptoethanol 980 to μl (20 sample buffer) -mercaptoethanol 950 μl (50 μl to sample buffer)

-mercaptoethanol (added fresh) O O O 2 2 2 .5 M Tris-HCl, pH 6.8 M Tris-HCl, .5 .0% Bromophenol blue blue Bromophenol .0% 1 diH Sample Buffers 2x SDS-PAGE (Laemmli, 30 ml) (catalog #1610737) glycerol, SDS, 25% pH 2% 6.8, mM Tris-HCl, 62.5 b 5% blue, bromophenol 0.01% Glycerol50% (added fresh) (added pH 6.8 M Tris-HCl, 0.5 SDS 10% Add b before use. Add b use. before 2x Native (30 PAGE ml) (catalog #1610738) glycerol, pH 40% 6.8, mM Tris-HCl, 62.5 blue bromophenol 0.01% 0 Glycerol50% blue Bromophenol 1.0% 2x Tricine (30 ml) (catalog #1610739) 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, 1.0 Glycerol100% SDS 10% G-250 blue Coomassie diH diH

l l 1 m 5 m 0 ml 0 ml 0 ml 0 ml .10 g .10 .40 g .40 .00 g 5 8 6 9 0 7.23 g 7.23 7.60 g 7.60 2 0.00 g 0.00 6 2 8 1 o 150 ml o 150 o 100 ml o 100 o 100 ml o 100 o 300 ml t t t

t

-Butanol , 1 L) , 1 L)

O O O O O O O O O 2 2 2 2 2 2 2 2 2 Butanol atalog#1610799 atalog #161-0125, 37.5:1 acrylamide/bis powder acrylamide/bis 37.5:1 atalog #161-0125, crylamide ml) g/100 (29.2 iH - c catalog #1610700) catalog #1610416) catalog #1610798 Store at 4ºC. 0.5 M Tris-HCl, pH 6.8 ( Buffer Formulations Gel Casting Reagents Acrylamide/Bis 2.67% (30% T, C) A N'N'-bis-methylene-acrylamide diH Tris base (18.15 g/100 ml) g/100 base (18.15 Tris d (w/v)10% APS (fresh daily) ( persulfate Ammonium diH Filter and store at 4ºC in the dark (30 days). Premade alternatives: C 30% acrylamide/bis solutionCatalog #161-0158, 1.5 M Tris-HCl, pH 8.8 ml) (150 ( 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) ( SDS diH Dissolve with gentle stirring n Water-Saturated Combine in a bottle and shake. Use the phase top only. Store at room temperature. diH n

0 ml 0 ml 0 ml .0 ml .0 ml .0 ml .0 ml .6 .50 g .50 .50 g .50 .20 g .20 g .20 .60 g .60 g .00 g 4 8 8 1 1 1 2 7 .92 ml.92 .08 ml ml .08 1.00 g 1.00 2 0 0 0 0 0 0.00 g 0.00

2 0 2 o 50 ml o 50 ml o 45 ml 2 t t t o 100 ml o 100 ml o 100 t t

, 30 ml)

Ampholytes

®

O O O O O

2 2 2 2 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, 1% (w/v) DTT, DTT, (w/v) CHAPS, (w/v) 1% 4% M urea,M thiourea,7 atalog #1610737 cetone cetone -mercaptoethanol ml) DTT or 3% (0.4 immediately 0% Trichloroacetic0% DTT acid 0.2% in ice-cold (TCA), .2% DTT.2% in ice-cold acetone (–20°C) c diH Tris base Tris SDSSample Solubilization Buffer (50 ml) (pH mM Tris-HCl SDS, 9.5) 100 (w/v) 1% SDS diH DTT Titrate to pH 9.5 withTitrate pH 9.5 diluted to HCl. diH DTT A Acetone Store at –20°C. ml) (100 Solution Wash A Acetone Store at –20°C. Lysis Buffer (50 ml) 2 carrier (v/v) 2% ampholytes (pH 3–10) Urea Thiourea diH CHAPS Bio-Lyte acetone (–20°C) TCA Dissolve DTT Dissolve Store as 1–2 mlStore and aliquots as 1–2 add at –70°C b use. before ml) (100 Solution Precipitation Protein 2 0 25% Glycerol25% blue Bromophenol 1.0% SDS 10% diH SDS-PAGESample Buffer (2x, 8 ml) glycerol, SDS, 2% pH 25% 6.8, mM Tris-HCl, 62.5 blue bromophenol 0.01% pH 6.8 mM Tris-HCl, 0.5 (

g 1 g 1 0 ml 0 ml 44 g

.15 g .15 .10 g .10 .5 ml.5 .24 g o 1 L .20 g .20 .88 g .06 g .00 g .00 g 8 8 60 ml 00 ml t 2.11 g 2.11 0 0 2 1. 1 0 0 0 6 8 00 mg 8 1 ~

o 10 ml o 10 ml o 10 1 t t o 100 ml o 100 o 100 ml o 100 ml o 100 t t t

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 just pH with 6.8 to HCl.

iH .9% (w/v) sodium (w/v) .9% chloride mM phosphate in 10 5 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, mM NaCl, 5 mM EDTA, 150 pH5 mM 7.6, Tris-HCl catalog #1610416) catalog #1610404) catalog KH Sample Preparation Buffers Sample Preparation 1 M Tris-HCl, (100 ml) pH 7.6 base Tris H Deionized Buffer Formulations diH d Adjust pH to 7.6 with HCl. Adjust 7.6 pH to Sodium deoxycholate Sodium diH pH 7.6 1 M Tris-HCl, diH L) 1 (PBS, Buffered Saline Phosphate buffer, pH 7.4 NaCl Na with HCl or NaOH. Adjust 7.4 pH to diH diH SDS Bromophenol1.0% Blue ml) (10 blue Bromophenol SolubilizationRIPA Buffer (100 ml) EDTA X-100 NP-40 or Triton KCl 0.5 M Tris-HCl, pH 6.8 (100 ml) (catalog #1610799) base Tris Ad Store at 4°C. SDS ml)10% (10 ( diH 2 sodium deoxycholate, 1% X-100, Triton NP-40 or 1% 1% SDS 0.1% NaCl SDS 0 diH diH (

64 ElectrophoresisGuide

TABLE OF CONTENTS 67 Methods

o 1 L t 0.60 g 0.60 5.00 g 5.00 00 mg 900 ml o 10 ml o 10 2 1 6 t ~ o 250 ml o 250 t

O O O O 2 2 2 2 catalog #1610416) #1610404) catalog catalog #1610799) diH Adjust pH with 6.8 to HCl. diH blue Bromophenol 1.0% Bromophenol1.0% Blue ml) (10 ( Buffer Components Store at 4°C. SDS10% (250 ml) SDS ( diH Tris base Tris diH 0.5 M Tris-HCl, pH 6.8 L) (1 (

o 1 L o 1 L o 1 L t t t 0.30 g 0.30 0.30 g 0.30 0.00 g 0.00 0.00 g 0.00 21.10 g 21.10 44.10 g 44.10 44.10 g 44.10 1 1 79.20 g 79.20 3 3

1 1 1 1

O O O 2 2 2

ris base ris base Glycine (catalog #1610732)pH 8.3 SDS, 1% glycine, M 1.92 Tris, mM 250 Running Buffers 10x SDS-PAGE L) (1 T Glycine SDS diH Buffer Formulations SDS diH T Tricine Do not adjust 8.3). the pH (~pH diH Do not adjust the pH (~pH 8.3). Do not adjust 8.3). the pH (~pH 10x Native L) (1 PAGE (catalog #1610734) M glycine, pH 8.3 1.92 mM Tris, 250 base Tris Do not adjust 8.3). the pH (~pH L) (1 10x Tris-Tricine (catalog #1610744) SDS, pH 8.3 1% 1 M Tricine, 1 M Tris,

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69 69 Troubleshooting

PartIII 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 ElectrophoresisGuide

TABLE OF CONTENTS

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 theentire range). molecular weight Fig. 1. Schematic of protein protein of Schematic 1. Fig. SDS-PAGE. during migration TIPS

ts under gasket of notch at top lution lution et gasket with running buffer Ensure that edge top short of plate fi Ensure that top of short plate gasket green the touches ll inner and outer buffer outer inner chambers and ll heck buffer protocol and heck buffer composition heck solutions or weights heck buffer composition and type heck electrodes and connections and electrodes heck ecreasevoltage 25–50% by sampleesalt se different %T se correct %C se correct %C se electrophoresis-grade reagents electrophoresis-grade se educe APS and TEMED by emove tape ipet sample into well slowly. Do slowly. well into sample ipet efore use efore 0%; degas0%; ill buffer chamber with 5% each 5% nd type oncentrate if necessary  unning bufferunning o ensure wells are completely ncrease APS and TEMED by nclude 10% glycerolnclude in 10% So So 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 C C c D C a D W R 2 I 5 U U U C U I sample make it to denser buffer surrounding than P  well, squirt quickly sample into not R t covered C • • b F r Fi

ffer chamber

w %T e electrode/companion assembly el inhibition; polymerization ross-linker high too is concentration high too unning buffer concentrated too unning or reservoir buffer ample not dense enough ipetting, loading error xcess catalysts; sample in salt xcessive oor quality acrylamide or bis lectrical disconnection disconnection lectrical olymerization min time <10 uffer chamber el cassette not removed oltage high too r type used nd gel temperature high; too omposition or type me >2 hr me >2 ape at the bottom precast of oo little cross-linker oo dilute ncorrect running buffer ncomplete gasket seal nsufficient buffer in inner nsufficient buffer outer in mproper assembly gel of into ncorrect running buffer concentration R a i o R t V I c E I E p G ti Lo P T B C S P T g b E th Cause Cause I I bu I (contd.)

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

Electrophoresis Electrophoresis

min 2-D Cleanup Kit) Cleanup to 2-D ™ aves during cell lysis and rotein solubilization iminish carbohydrate content carbohydrate iminish f stacking APS gel 0.06% to f stacking APS gel 0.06% to nd 0.12% TEMED nd 0.12% TEMED nd 0.12% d Tris base untild Tris buffer turns lution lution p (ReadyPrep w d 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 o a Prepare fresh catalyst solution Increase catalyst concentration o a Wash gasket if it is dirty Replace flawed wornor out casting stand gaskets ast at room temperature, inse or wipe off powder residue se electrophoresis-grade reagents electrophoresis-grade se se 0.05% APS and 0.05% TEMED APS and 0.05% se0.05% repare fresh APS nsure plates are free flaws of nsure plates are aligned correctly efore each use rior casting to stacking gel lue again

 

So So Ad E E

C  necessary if plates glass warming U P p • • immediately solution monomer Degas • • R b U 10–15 solutions monomer Degas • • • • • b

onomer solution not degassed not solution onomer ld APS hipped glass plates asting stand gasket dirty, igh DNA or carbohydrate content ample buffer is acidic too pacer plate and short plate not level oor quality acrylamide or bis owder residue has built up at ivot point pressure of cams ailure degas to emperature low too oo little or much too APS or TEMED awed, or worn out

ncorrect catalyst concentration ncorrect catalyst used oxygen inhibits polymerization)

S C S C

T P O ( I I M P p T F Cause fl Cause H

el does not polymerize

Problem Leaking during handcasting Laemmli sample buffer turns Problem yellow

Webbing; excess acrylamide Pressure cams on casting frame are difficultto close or make noise when closed G

behind the comb Poor well formation Sample very viscous Gel Casting and Sample Loading Sample and Casting Gel Sample Preparation

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TABLE OF CONTENTS 73 Troubleshooting

ck buffer composition and and bufferck composition iluting 5x or 10x stockiluting or 10x 5x uring stackinguring ot mixedot well, or buffer in upper .5% or 5% T or 5% .5% hamber concentrated too emove residual acrylamide residual emove d sample buffers sample d horough mixing, especially mixing, whenhorough nimize time between sample between time nimize lution inimize salts, detergents, and d t Increase stacking of %T gel to 4 Increase current 25% by d rate polymerization Decrease carefully gels Overlay Rinse wells after removing comb to r Use electrophoresis-grade reagents reagents electrophoresis-grade Use conditions polymerization Check buffer bufferCheck composition; n c ensuring buffer, new Prepare ll inner and outer buffer outer inner and ll o not exceed recommended se correct voltage correct se se same buffer in samples as se external cooling during run or emove salts from sample by repare fresh solutions ower setting V from 200 150 V to ialysis or desalting column prior column desalting or ialysis ilution instructions r fill lower chamberto within 1 cm f top of short of f top plate oad less protein oad re completely covered startup power and pplication hambers ensure to that wells olvents in sample preparation

unning conditions. Decrease power Decrease conditions. unning un more slowly more un o sample preparation n gel M s an U R U i Che d Mi a p o o Fi c a L d t D r

So • • • • • • • P U r • •

at of gelat of s-acrylamide, incomplete incomplete s-acrylamide, verloaded proteins verloaded ld SDS or sample buffer rong formulation el temperature high too iffusion prior to turning on current on turning iffusion to prior through iffusion migration during neven gel interface ample preparation/buffer issues xcess salt in samples xcess heating gel; of center of ower conditions excessive oor qualityoor acrylamide or el runs hotter than either end tacking gel

ncorrect running conditions running ncorrect onic strength sample of lower than nsufficient buffer nsufficientbuffer sample or

S I E I th I w D D I O P

bi P s U Cause polymerization O G E g

lateral band spreading band lateral gel lane Skewed or distorted bands,

within bands frowning or Smiling

Problem bands Diffuse broad or

Bands“smile” across gel, band pattern curves upward at both sides gel of Evaluation of Separationof Evaluation

ck instrument manual for manual instrument ck manual instrument ck estaining solution for min ≥30 els only edges by nder notch on gasket on notch nder olution are exposed open to air ecommended in protocol in ecommended lution lution n staining solutions as Restrict durationincubation of i r Wash gel in water or respective d thoroughly trays Clean staining Limit time that gels and staining s handle and gloves dust-free Use g Ensure that U-shaped electrode core gasket is clean, free cuts, of and lubricated with buffer Ensure that short plate is u lean staining trays and other lean staining trays and other se high-purity water and reagents epeat staining protocol with fresh tain with another method to eep buffer level below of top gitate gel during staining during gel gitate oubleshooting, or contact lassware cleaner lassware lassware cleaner lassware ollow recommendations for stain for recommendations ollow quipment with laboratory laboratory with quipment quipment with laboratory laboratory with quipment onfirm there is protein pacer plate taining solution taining ransfer gel water for rehydration to olume (appropriate to gel size) gel to (appropriate olume

or staining or ncrease staining time staining ncrease maging instrument manufacturer instrument maging v I R s C So So C e g F e g S c Che tr i Che s • • U f • • • • • K A T



re used el dehydrated o protein in gel irty equipment or staining trays irty staining trays pper buffer chamber overfilled euse staining of solution eagent impurities articulate material from reagents, taining tray, dust,taining or gloves tray, oo much time in staining solution

nsufficienttime staining nsufficient stain volume nsufficientstain ncorrect imaging parameters imaging ncorrect maging system malfunctioning malfunctioning system maging mproper assemblymproper nsufficient shakingstaining during I R D D I T N I I we I R P Cause Cause U I G s

Tetra Cell) Tetra (contd.) ®

image

High or uneven or High background staining background

Poor staining sensitivity

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

Uneven staining Uneven

Speckles or blotches in gel

(Mini-PROTEAN Gel shrinkage Total Protein Staining Protein Total Electrophoresis Electrophoresis

72 ElectrophoresisGuide

TABLE OF CONTENTS 75 Troubleshooting inimize streaking inimize rotein in sample (fractionate) articulates prior to sample loading sample to prior articulates lution Dilute sampleDilute predominant remove Selectively p Reduce to voltage 25% by m Centrifuge samples remove to p Dilute sample in sample buffer se 5% BME or 1% DTT BME orse 1% 5% or a differentse SDS-PAGE uffer system in native PAGE r IEF

So • • • • • U U b o

mple precipitation precipitation mple ore negative than pH of verloaded samples oncentration reducing of andsinterest of may be neutral l of bandsl of pH must units be ~2 r positively charged in buffer used; gent low too unning bufferunning

O Sa C a B o p m r Cause

(contd.)

Vertical streaking Problem

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

74 ElectrophoresisGuide

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 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 or PVDF may or may not sieve the macromolecules

Antibody Immunoglobulin; protein produced in response to an antigen, 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) R educing 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 T echnique 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 A ntibody; 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 P ortion 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 Monomer Unit that makes up a polymer (acrylamide is a monomer that is polymerized into SDS), tracking dye, and glycerol polyacrylamide) SDS-PAGE Separation of molecules by molecular weight in a polyacrylamide gel matrix in the Mini-PROTEAN® Family of Bio-Rad products used for mini-format vertical electrophoresis; presence of a denaturing detergent, (SDS). SDS denatures TABLE CONTENTS OF Cells and Gels includes the Mini-PROTEAN Tetra and Mini-PROTEAN® 3 Dodeca™ Cells, and polypeptides and binds to proteins at a constant charge-to-mass-ratio. In a sieving Mini-PROTEAN Precast Gels polyacrylamide gel, the rate at which the resulting SDS-coated proteins migrate in Native PAGE Version of PAGE that retains native protein configuration, performed in the absence the gel is relative only to their size and not to their charge or shape of SDS and other denaturing agents Secondary antibody Reporter antibody that binds to a primary antibody; used to facilitate detection Ohm’s Law D escribes the mutual dependence of three electrical parameters (V, volts; Sodium dodecyl Anionic detergent that denatures proteins and binds to polypeptides in a constant I, ampere; R, ohm): V = I x R sulfate (SDS) weight ratio of 1:4 (SDS:polypeptide) PAGE Polyacrylamide gel electrophoresis, a common method of separating proteins Spacers Small blocks set between the two glass plates at the sides of a gel cassette, Polyacrylamide A nticonvective sieving matrix used in gel electrophoresis. Polyacrylamide gels are which create a space between the glass plates in which to pour the slab gel cast using mixtures of acrylamide monomers with a cross-linking reagent, usually monomer solution N,N'-methylenebisacrylamide (bis), both solubilized in buffer Stacking gel P ortion of a discontinuous electrophoresis gel that concentrates the components of Polyacrylamide gel Electrophoresis technique that uses polyacrylamide as the separation medium the sample to create a very thin starting zone; bands are then separated from each electrophoresis (PAGE) other in the resolving gel

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

80 81 Electrophoresis Guide Appendices

Total protein stain R eagent 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 Total protein In total protein normalization, the abundance of the target protein is normalized Berkelman T (2008). Quantitation of protein in samples prepared for 2-D electrophoresis. Methods Mol Biol 424, 43–49. normalization to the total amount of protein in each lane, removing variations associated with Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye normalization against a single protein binding. Anal Biochem 72, 248–254. Cañas B et al. (2007). Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153, 235–258. Tricine  used in SDS-PAGE as a buffer component to replace glycine Chen Y et al. (2008). Sample preparation. J Chromatogr A 1184, 191–219. and improve resolution of small (down to 1–5 kD) proteins Damerval C et al. (1988). Two-dimensional electrophoresis in plant biology. Advances in Electrophoresis 2, 236–340. Tris Organic component of buffer solutions that has an effective pH range of 7.0–9.2; Drews O et al. (2004). Setting up standards and a reference map for the alkaline of the Gram-positive bacterium Lactococcus lactis. tris(hydroxymethyl) aminomethane Proteomics 4, 1293–1304. Evans DR et al. (2009). Concentration of proteins and removal of solutes. Methods Enzymol 463, 97–120. Transfer Immobilization of proteins or other molecules onto a membrane by electrophoretic Goldberg S (2008). Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol Biol 424, 3–22. or passive means Harder A et al. (1999). Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Electrophoresis 20, 826–829. Triton X-100 Nonionic detergent widely used for protein solubilization (for IEF and 2-D Huber LA et al. (2003). Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res. 92, 962–968. electrophoresis) Lowry OH et al. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. Tween 20 Nonionic detergent; used in blot detection procedures as a blocking reagent or in Luche S et al. (2003). Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. wash buffers to minimize nonspecific binding and background Proteomics 3, 249–253. Noble JE and Bailey MJ (2009). Quantitation of protein. Methods Enzymol 463, 73–95. Unstained standards Mixture of molecular weight marker proteins that do not have covalently attached Poetsch A and Wolters D (2008). Bacterial membrane proteomics. Proteomics 8, 4100–4122. dye molecules; the bands are invisible during electrophoresis and transfer, but are Posch A et al. (2006). Tools for sample preparation and prefractionation in two-dimensional gel electrophoresis. In Separation Methods in useful for molecular weight determination Proteomics, Smejkal GB ed. (Boca Raton: CRC Press), 107–133. Rabilloud T (1996). Solubilization of proteins for electrophoretic analyses. Electrophoresis 17, 813–829. TABLE CONTENTS OF Urea Chaotrope usually included at rather high concentrations (9.5 M) in sample Rhodes DG and Laue TM (2009). Determination of protein purity. Methods Enzymol 463, 677–689. solubilization buffers for denaturing IEF and 2-D PAGE Sapan CV et al. (1999). Colorimetric protein assay techniques. Biotechnol Appl Biochem 29, 99–108. Western blotting Immobilization of proteins onto a membrane and subsequent detection by Smith PK et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85. protein-specific binding and detection reagents Vuillard L et al. (1995). Enhancing protein solubilization with nondetergent sulfobetaines. Electrophoresis 16, 295-297.

Zymogram PAGE Electrophoresis technique used to detect and characterize collagenases and Electrophoresis other proteases within the gel. Gels are cast with gelatin or casein, which Andrews AT (1986). Electrophoresis: theory, techniques and biochemical and clinical applications (New York: Oxford University Press). acts as a substrate for the enzymes that are separated in the gel under Bjellqvist B et al. (1982). Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys nonreducing conditions 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.

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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. 6385 Biologics Analysis Workflow Brochure Fernandez-Patron C et al. (1992). Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of 1069 Colorimetric Protein Assays unmodified proteins. Biotechniques 12, 564-573. 2414 The Little Book of Standards Gottlieb M and Chavko M (1987). Silver staining of native and denatured eucaryotic DNA in agarose gels. Anal Biochem 165, 33-37. 2998 Protein Standards Application Guide 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. 2317 Ready-to-Run Buffers and Solutions Brochure ® Lee C et al. (1987). Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 166, 308-312. 5535 Mini-PROTEAN Tetra Cell Brochure ® ™ Merril CR (1987). Detection of proteins separated by electrophoresis. Adv Electrophoresis 1, 111–139. 5871 Mini-PROTEAN TGX Precast Gels Product Information Sheet ™ Merril CR et al. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 2710 Criterion Precast Gel System Brochure 211, 1437–1438. 2911 Criterion XT Precast Gels Product Information Sheet Miller I et al. (2006). Protein stains for proteomic applications: which, when, why? Proteomics, 6, 5385–5408. 5974 Criterion TGX Stain-Free 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 1760 PROTEAN ® II xi and XL Cells Product Information Sheet nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262. 6371 Electrophoresis Power Supplies Brochure Oakley BR et al. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105, 361–363. 2423 Bio-Safe™ Coomassie Stain Brochure Rabilloud T et al. (1994). Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40, 57–75. 5346 Flamingo™ Fluorescent Gel Stain Product Information Sheet Simpson RJ (2010). Rapid coomassie blue staining of protein gels. Cold Spring Harb Protoc, pdb prot5413. 5900 Oriole™ Fluorescent Gel Stain Product Information Sheet Sinha P et al. (2001). A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of 3096 Expression Proteomics Brochure proteins after two-dimensional electrophoresis. Proteomics 1, 835-840. Steinberg TH (2009). Protein gel staining methods: an introduction and overview. Methods Enzymol 463, 541–563.

Steinberg TH et al. (2003). Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3, 1128–1144. Instruction Manuals Westermeier R and Marouga R (2005). Protein detection methods in proteomics research. Biosci Rep 25, 19–32. Bulletin Title Yan JX et al. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization 10007296 Mini-PROTEAN Tetra Cell

TABLE CONTENTS OF and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672. 1658100 Mini-PROTEAN Precast Gels 4006183 Criterion Cell Bio-Rad Bulletins 4006213 PowerPac Basic Power Supply Technical Notes 4006222 PowerPac HC Power Supply Bulletin Title 4006223 PowerPac Universal Power Supply 6387 Biologics Analysis Workflow Comparability Study 4110001 Criterion Gel Application Guide 2651 2-D Electrophoresis for Proteomics: A Methods and Product Manual 4110130 Criterion XT Precast Gels 2895 Protein Blotting Guide, A Guide to Transfer and Detection 4110065 Quick Start™ Bradford Protein Assay 1156 Acrylamide Polymerization – a Practical Approach 4110107 RC DC™ Protein Assay 1909 Modification of Bio-Rad DC™ Protein Assay for Use with Thiols 4000126-14 The Discovery Series: Quantity One® 1-D Analysis Software 6001 Rapid Validation of Purified Proteins Using Criterion™ Stain Free™ Gels LIT33 Bio-Rad Protein Assay 5910 Mini-PROTEAN® TGX™ Precast Gel: A Gel for SDS-PAGE with Improved Stability — Comparison with Standard Laemmli Gels LIT448 DC Protein Assay 5911 Mini-PROTEAN TGX Precast Gel: A Versatile and Robust Laemmli-Like Precast Gel for SDS-PAGE 5932 Ready Gel® to Mini-PROTEAN TGX Precast Gels Catalog Number Conversion Chart 5934 NuPAGE Bis-Tris Precast Gels (MOPS Buffer) to Mini-PROTEAN TGX Precast Gels Catalog Number Conversion Chart 3133 Molecular Weight Determination by SDS-PAGE 3144 Using Precision Plus Protein™ Standards to Determine Molecular Weight 5956 Precision Plus Protein Dual Xtra Standards – New Protein Standards with an Extended Range from 2 to 250 kD 5763 Molecular Weight Estimation Using Precision Plus Protein™ WesternC™ Standards on Criterion Tris-HCl and Criterion™ XT Bis-Tris Gels 5576 Molecular Weight Estimation and Quantitation of Protein Samples Using Precision Plus Protein WesternC Standards, 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

84 85 Electrophoresis Guide Appendices

Ordering Information Electrophoresis Instrumentation Catalog # Description Catalog # Description Catalog # Description Catalog # Description

Mini-PROTEAN® Tetra Cells and Systems 1658033 Mini-PROTEAN Tetra Cell, Mini Trans-Blot PROTEAN® Plus Dodeca™ Cells and Systems Protein Assay Kits and Instruments 1658000 M ini-PROTEAN Tetra Cell, 10-well, 0.75 mm Module, and PowerPac Basic Power Supply, 1654150 P ROTEAN Plus Dodeca Cell, 100/120 V, includes 5000001 Bio-Rad Protein Assay Kit I, includes 450 ml dye thickness; 4-gel system includes 5 combs, 5 sets includes 165-8001, 170-3935, and 164-5050 electrophoresis buffer tank with built-in ceramic reagent concentrate, bovine b-globulin standard; of glass plates, 2 casting stands, 4 casting frames, 1658034 M ini-PROTEAN Tetra Cell for Mini Precast Gels, cooling core, lid, buffer recirculation pump with sufficient for 440 standard assays or 2,200 sample loading guide, electrode assembly, Mini Trans-Blot Module, and PowerPac Basic tubing, 2 gel releasers microplate assays companion running module, tank, lid with power Power Supply, includes 1658004, 1703935, 1654140 PROTEAN Plus Dodeca Cell (100/120 V) and 5000002 Bio-Rad Protein Assay Kit II, includes 450 ml cables, mini cell buffer dam and 1645050 PowerPac HC Power Supply, includes 1654150 dye reagent concentrate, 1658001 Mini-PROTEAN Tetra Cell, 10-well, 1.0 mm 1658035 M ini-PROTEAN Tetra Cell, Mini Trans-Blot and 1645052 standard; sufficient for 440 standard assays or thickness; 4-gel system includes 5 combs, 5 sets Module, and PowerPac HC Power Supply, 1654142 PROTEAN Plus Dodeca Cell (100/120 V) and 2,200 microplate assays of glass plates, 2 casting stands, 4 casting frames, includes 1658001, 1703935, and 1645052 PowerPac Universal Power Supply, includes 5000111 DC™ Protein Assay Kit I, includes 250 ml alkaline sample loading guide, electrode assembly, 1658036 M ini-PROTEAN Tetra Cell for Mini Precast Gels, 1654150 and 1645070 copper tartrate solution, 2 L dilute Folin reagent, companion running module, tank, lid with power Mini Trans-Blot Module, and PowerPac HC Power 1654144 P ROTEAN Plus Dodeca Cell (100/120 V), 5 ml surfactant solution, bovine b-globulin standard; cables, mini cell buffer dam Supply, includes 1658004, 1703935, and 1645052 Trans-Blot Plus Cell, and PowerPac Universal Power sufficient for 450 standard assays 1658002 Mini-PROTEAN Tetra Cell, 2 10-well, 0.75 mm Mini-PROTEAN® Dodeca™ Cells and Systems Supply, includes 1654150, 1703990, and 1645070 5000112 DC Protein Assay Kit II, includes 250 ml alkaline thickness; 2-gel system includes 5 combs, 5 sets of 1654100 Mini-PROTEAN 3 Dodeca Cell, includes 1655134 PROTEAN Plus Dodeca Cell (100/120 V) and copper tartrate solution, 2 L dilute Folin reagent, 5 ml glass plates, casting stand, 2 casting frames, sample electrophoresis tank with built-in cooling coil, lid with Two 6-Row AnyGel Stands, includes 1654150 surfactant solution, bovine serum albumin standard; loading guide, electrode assembly, tank, lid with power cables, 6 electrophoresis clamping frames, and two 1655131 sufficient for 450 standard assays power cables, mini cell buffer dam ™ 2 buffer dams, drain line, 2 gel releasers 1654151 P ROTEAN Plus Dodeca Cell, 220/240 V, includes 5000120 RC DC Protein Assay Reagents Package, 1658003 Mini-PROTEAN Tetra Cell, 10-well, 1.0 mm 1654101 Mini-PROTEAN 3 Dodeca Cell with Multi-Casting electrophoresis buffer tank with built-in ceramic includes 250 ml alkaline copper tartrate solution, thickness; 2-gel system includes 5 combs, 5 sets of Chamber, same as 165-4100 with multi-casting cooling core, lid, buffer recirculation pump with 2 L dilute Folin reagent, 5 ml surfactant solution; glass plates, casting stand, 2 casting frames, sample chamber, 15 separation sheets, 8 acrylic blocks, tubing, 2 gel releasers sufficient for 450 standard assays loading guide, electrode assembly, tank, lid with tapered luer connector, stopcock valve 5000121 RC DC Protein Assay Kit I, includes RC reagents power cables, mini cell buffer dam 1654141 PROTEAN Plus Dodeca Cell (220/240 V) and Criterion™ Cells and Systems PowerPac HC Power Supply, includes 1654151 package, DC reagents package, bovine b-globulin 1658004 M ini-PROTEAN Tetra Cell for Mini Precast Gels, standard; sufficient for 450 standard assays

TABLE CONTENTS OF 1656001 Criterion Cell, includes buffer tank, lid with power and 1645052 4-gel system includes electrode assembly, clamping cables, 3 sample loading guides (12 + 2-well, 5000122 RC DC Protein Assay Kit II, includes RC reagents frame, companion module, tank, lid with power 1654143 P ROTEAN Plus Dodeca Cell (220/240 V) and 18-well, 26-well) package, DC reagents package, bovine serum cables, mini cell buffer dam PowerPac Universal Power Supply, includes 1656019 Criterion Cell and PowerPac Basic Power Supply, 1654151 and 1645070 albumin standard; sufficient for 450 standard assays 1658005 Mini-PROTEAN Tetra Cell for Mini Precast Gels, 100–120/220–240 V, includes 1656001 and 1645050 5000201 Quick Start™ Bradford Protein Assay Kit 1, 2-gel system includes electrode assembly, clamping 1654145 P ROTEAN Plus Dodeca Cell (220/240 V), includes 1x dye reagent (1 L), bovine serum albumin frame, tank, lid with power cables, mini cell buffer dam Criterion™ Dodeca™ Cells and Systems Trans-Blot Plus Cell, and PowerPac Universal Power 1654130 Cr iterion Dodeca Cell, includes electrophoresis Supply, includes 1654151, 1703990, and 1645070 standard (5 x 2 mg/ml); sufficient for 200 standard 1658006 M ini-PROTEAN Tetra Cell, 10-well, 1.5 mm buffer tank with built-in cooling coil, lid with assays or 4,000 microplate assays thickness; 4-gel system includes 5 combs, 5 sets 1655135 P ROTEAN Plus Dodeca Cell (220/240 V) and power cables 5000202 Quick Start Bradford Protein Assay Kit 2, of glass plates, 2 casting stands, 4 casting frames, Two 6-Row AnyGel Stands, includes 1654151 includes 1x dye reagent (1 L), bovine serum albumin sample loading guide, electrode assembly, 1654138 Criterion Dodeca Cell and PowerPac HC Power and two 1655131 standard set (2 sets of 7 concentration standards, companion running module, tank, lid with power Supply, includes 1654130 and 1645052 Power Supplies 0.125–2.0 mg/ml, 2 ml) cables, mini cell buffer dam 1654139 Criterion Dodeca Cell and PowerPac Universal 1645050 PowerPac Basic Power Supply, 100–120/220–240 V 5000203 Quick Start Bradford Protein Assay Kit 3, 1658007 Mini-PROTEAN Tetra Cell, 10-well, 1.5 mm Power Supply, includes 1654130 and 1645070 1645052 PowerPac HC Power Supply, 100–120/220–240 V includes 1x dye reagent (1 L), bovine b-globulin thickness; 2-gel system includes 5 combs, 5 sets of ™ 1655133 Criterion Dodeca Cell and 6-Row AnyGel Stand, 1645056 PowerPac HV Power Supply, 100–120/220–240 V standard (5 x 2 mg/ml) glass plates, casting stand, 2 casting frames, sample includes 1654130 and 1655131 1645070 PowerPac Universal Power Supply, loading guide, electrode assembly, tank, lid with 5000204 Quick Start Bradford Protein Assay Kit 4, PROTEAN® II xi Cells 100–120/220–240 V power cables, mini cell buffer dam includes 1x dye reagent (1 L), bovine b-globulin 1651801 PROTEAN II xi Cell, 16 cm, without spacers and combs Sample Preparation Kits standard set (2 sets of 7 concentration standards, 1658025 Mini-PROTEAN Tetra Cell and PowerPac™ Basic 1651802 PROTEAN II xi Cell, 16 cm, 1.5 mm spacers (4), 1632141 MicroRotofor™ Cell Lysis Kit (Mammal), 15 preps, 0.125–2.0 mg/ml, 2 ml) Power Supply, includes 1658001 and 1645050 includes 50 ml protein solubilization buffer (PSB), 15-well combs (2) 1702502 Standard , 1–3.5 ml, quartz 1658026 Mini-PROTEAN Tetra Cell and PowerPac™ ReadyPrep™ mini grinders (2 packs of 10 each) 1651803 P ROTEAN II xi Cell, 16 cm, 1.0 mm spacers (4), ™ Universal Power Supply, includes 1658001 1702511 trUView Cuvettes, pack of 100, individually packaged, 15-well combs (2) 1632142 MicroRotofor Cell Lysis Kit (Plant), 10 preps, disposable DNase- and RNase-free cuvettes and 1645070 includes 50 ml protein solubilization buffer (PSB), 1651804 PROTEAN II xi Cell, 16 cm, 0.75 mm spacers (4), 1658027 M ini-PROTEAN Tetra Cell and PowerPac™ HC ReadyPrep 2-D cleanup kit (50 reaction size) Sample Preparation Buffers and Reagents 15-well combs (2) 1610747 4x Laemmli Sample Buffer, 10 ml Power Supply, includes 1658001 and 1645052 1632143 MicroRotofor Cell Lysis Kit (Yeast), 15 preps, 1651811 P ROTEAN II xi Cell, 20 cm, without spacers 1610737 Laemmli Sample Buffer, 30 ml 1658028 Mini-PROTEAN Tetra Cell and PowerPac™ HV includes 50 ml protein solubilization buffer (PSB), and combs Power Supply, includes 1658001 and 1645056 15 ml yeast suspension buffer, 2 x 0.5 ml lyticase 1610738 Native Sample Buffer, 30 ml 1651812 P ROTEAN II xi Cell, 20 cm, 1.5 mm spacers (4), 1610739 Tricine Sample Buffer, 30 ml 1658029 Mini-PROTEAN Tetra Cell and Mini Trans-Blot® (1.5 U/µl) 15-well combs (2) Module, includes 1658001 and 1703935 1632144 MicroRotofor Cell Lysis Kit (Bacteria), 15 preps, 1610763 IEF Sample Buffer, 30 ml 1651813 PROTEAN II xi Cell, 20 cm, 1.0 mm spacers (4), 1610791 XT Sample Buffer, 4x, 10 ml 1658030 M ini-PROTEAN Tetra Cell for Mini Precast Gels includes 50 ml protein solubilization buffer (PSB), 15-well combs (2) and Mini Trans-Blot Module, includes 1658004 25 ml bacteria suspension buffer, 1 ml lysozyme 1610792 XR Reducing Agent, 1 ml and 1703935 1651814 P ROTEAN II xi Cell, 20 cm, 0.75 mm spacers (4), (1,500 U/µl) 1610719 Tris, 1 kg 15-well combs (2) 1632140 ReadyPrep 2-D Cleanup Kit, 5 preps 1610718 Glycine, 1 kg 7326221 Micro Bio-Spin™ 6 Columns, includes 25 columns 1610301 SDS, 100 g in Tris buffer, 50 collection tubes 1610416 SDS Solution, 10% (w/v), 250 ml 7326227 Bio-Spin® 6 Columns, includes 25 columns 1662404 10% Tween 20, 5 ml in Tris buffer, 50 collection tubes 1610710 2-Mercaptoethanol, 25 ml 7326228 Bio-Spin 6 Columns, includes 100 columns 1610611 Dithiothreitol, 5 g in Tris buffer, 200 collection tubes 1610404 Bromophenol Blue, 10 g 1610730 Urea, 250 g

86 87 Electrophoresis Guide Appendices

Catalog # Description Catalog # Description Precast Gels Protein Standards Running Buffers and Reagents Description Recombinant Prestained Protein Standards 1610732 10x Tris/Glycine/SDS, 1 L 1610393 Precision Plus Protein All Blue Standards 10-Gels/Box 1610734 10x Tris/Glycine, 1 L Value Pack, 5 x 500 µl 1610744 10x Tris/Tricine/SDS, 1 L 10-Well 10-Well 15-Well IPG Well 12-Well 1610373 Precision Plus Protein All Blue Standards, 500 µl 30 µl 50 µl 15 µl 7cm IPG Strip 20 µl 1610788 XT MOPS Running Buffer, 20x, 500 ml 1610394 Precision Plus Protein Dual Color Standards 1610789 XT MES Running Buffer, 20x, 500 ml Mini-PROTEAN® TGX™ Resolving Gels Value Pack, 5 x 500 µl 1610790 XT Tricine Running Buffer, 20x, 500 ml 7.5% 4561023 4561024 4561026 4561021 4561025 1610374 Precision Plus Protein Dual Color Standards, 10% 4561033 4561034 4561036 4561031 4561035 1610793 X T MOPS Buffer Kit, includes 500 ml 20x XT MOPS 500 µl 12% 4561043 4561044 4561046 4561041 4561045 running buffer, 10 ml 4x XT sample buffer, 1 ml 18% 4561073 4561074 4561076 4561071 4561075 1610397 Precision Plus Protein Dual Xtra Standards 20x XT reducing agent 4–15% 4561083 4561084 4561086 4561081 4561085 Value Pack, 5 x 500 µl 1610796 X T MES Buffer Kit, includes 500 ml 20x XT MOPS 4–20% 4561093 4561094 4561096 4561091 4561095 1610377 Precision Plus Protein Dual Xtra Standards, running buffer, 10 ml 4x XT sample buffer, 1 ml 8–16% 4561103 4561104 4561106 4561101 4561105 500 µl 20x XT reducing agent 10 –20% 4561113 4561114 4561116 4561111 4561115

1610395 Precision Plus Protein Kaleidoscope Standards 1610797 X T Tricine Buffer Kit, includes 500 ml 20x XT Any kD 4569033 4569034 4569036 4569031 4569035 Value Pack, 5 x 500 µl MOPS running buffer, 10 ml 4x XT sample buffer, Mini-PROTEAN® TGX Stain-Free™ Gels 1 ml 20x XT reducing agent 1610375 Precision Plus Protein Kaleidoscope Standards, 7.5% 4568023 4568024 4568026 4568021 4568025 500 µl 1610761 10x IEF Anode Buffer, 250 ml 10% 4568033 4568034 4568036 4568031 4568035 1610399 Precision Plus Protein WesternC Standards 1610762 10x IEF Cathode Buffer, 250 ml 12% 4568043 4568044 4568046 4568041 4568045 Value Pack, 5 x 250 µl 1610765 Zymogram Renaturation Buffer, 125 ml 4–15% 4568083 4568084 4568086 4568081 4568085 1610376 Precision Plus Protein WesternC Standards, 1610766 Zymogram Development Buffer, 125 ml 4–20% 4568093 4568094 4568096 4568091 4568095 8–16% 4568103 4568104 4568106 4568101 4568105 250 µl 1610729 EDTA, 500 g Any kD 4568123 4568124 4568126 4568121 4568125 1610398 Precision Plus Protein WesternC 1610718 Glycine, 1 kg Mini-PROTEAN Tris-Tricine Gels (Pack of 2) (Standards + HRP) Value Pack, 5 x 250 µl 1610713 Tricine, 500 g 16.5% Resolving Gel 4563063 4563064 4563066 — 4563065* 1610385 Precision Plus Protein WesternC 1610719 Tris, 1 kg 10–20% Resolving Gel 4563113 4563114 4563116* — 4563115* TABLE CONTENTS OF (Standards + HRP), 250 µl Gel Casting Buffers and Reagents Mini-PROTEAN TBE Gels (Pack of 2) Recombinant Unstained Protein Standards 1615100 SDS-PAGE Reagent Starter Kit, includes 1610396 Precision Plus Protein Unstained Standards 100 g acrylamide, 5 g bis, 5 ml TEMED, 5% TBE Gel 4565013 4565014* 4565016 — 4565015* Value Pack, 5 x 1000 µl 10 g ammonium persulfate Mini-PROTEAN TBE-Urea Gels (Pack of 2) 1610363 Precision Plus Protein Unstained Standards, 1610100 Acrylamide, 99.9%, 100 g 10% TBE-Urea Gel 4566033* — 4566036* — — 1000 µl 1610120 Acrylamide/Bis Powder, 19:1, 30 g 15% TBE-Urea Gel 4566053* — 4566056* — 4566055* 1610122 Acrylamide/Bis Powder, 37.5:1, 30 g 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 1610140 40% Acrylamide Solution, 500 ml number for the corresponding ten pack. 1610144 40% Acrylamide/Bis Solution, 19:1, 500 ml 1610146 40% Acrylamide/Bis Solution, 29:1, 500 ml 1610148 40% Acrylamide/Bis Solution, 37.5:1, 500 ml 1610154 30% Acrylamide/Bis Solution, 19:1, 500 ml 1610156 30% Acrylamide/Bis Solution, 29:1, 500 ml 1610158 30% Acrylamide/Bis Solution, 37.5:1, 500 ml 1610200 Bis Crosslinker, 5 g 1610800 TEMED, 5 ml 1610798 Resolving Gel Buffer, 1.5 M tris-HCl, pH 8.8, 1 L 1610700 Ammonium Persulfate (APS), 10 g 1610799 Stacking Gel Buffer, 0.5 M tris-HCl, pH 6.8, 1 L

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 bio-rad.com for a complete listing of accessories, 12003153 ChemiDoc™ Imaging System, gel and blot imaging and analysis system, includes internal Criterion™ TGX™ Gels** including available empty gel cassettes and glass plates, spacers, 7.5% 5671023 5671024 5671025 — — combs, etc. computer, 12" touch-screen display, camera, Image Lab™ Touch Software, Image Lab Software, 10% 5671033 5671034 5671035 — — 1655131 A nyGel Stand, 6-row, holds 6 PROTEAN® Gels Blot/UV/Stain-Free Sample Tray. Optional upgrade 12% 5671043 5671044 5671045 — — or 12 Criterion Gels 4–15% 5671083 5671084 5671085 5671082 5671081 path to ChemiDoc MP for fluorescence detection 1654131 AnyGel Stand, single-row, holds 1 PROTEAN Gel 4–20% 5671093 5671094 5671095 5671092 5671091 12003154 ChemiDoc MP Imaging System, gel and blot or 2 Criterion Gels 8–16% 5671103 5671104 5671105 5671102 5671101 imaging and analysis system, includes internal Any kD 5671123 5671124 5671125 5671122 5671121 Total Protein Gel Stains computer, 12" touch-screen display, camera, Criterion™ TGX Stain-Free™ 1610803 Q C Colloidal Coomassie Solution Kit, 1 L, Image Lab Touch Software, Image Lab Software, Gels** ready-to-use, non-hazardous colloidal Coomassie Blot/UV/Stain-Free Sample Tray G-250 Stain for protein polyacrylamide gels 7.5% 5678023 5678024 5678025 — — 1707991 GS-900™ Calibrated Densitometry System, ™ 12% 5678043 5678044 5678045 — — 1610786 Bio-Safe Coomassie Stain, 1 L gel densitometry system, PC compatible, includes 18% 5678073 5678074 5678075 5678072 5678071 1610787 Bio-Safe Coomassie Stain, 5 L scanner, cables, Image Lab Software, optional 4–15% 5678083 5678084 5678085 5678082 5678081 21 CFR Part 11 and Installation Qualification/ 1610435 Coomassie Brilliant Blue R-250 Staining 4–20% 5678093 5678094 5678095 5678092 5678091 Operations Qualification 8–16% 5678103 5678104 5678105 5678102 5678101 Solutions Kit, includes 1 L Coomassie Brilliant Blue ™ Any kD 5678123 5678124 5678125 5678122 5678121 R-250 Staining Solution, 2 x 1 L Coomassie Brilliant 1708195 Gel Doc XR+ System with Image Lab Software, Blue R-250 Destaining Solution PC or Mac, includes darkroom, UV transilluminator, Criterion XT Bis-Tris Gels*** epi-white illumination, camera, cables, Image Lab 1610436 Coomassie Brilliant Blue R-250 Staining Solution, 1 L 10% Resolving Gel 3450111 3450112 3450113 — 3450115 Software 12% Resolving Gel 3450117 3450118 3450119 3450120† 3450121 1610438 Coomassie Brilliant Blue R-250 Destaining 1708270 Gel Doc EZ System with Image Lab Software, 4–12% Resolving Gel 3450123 3450124 3450125 3450126† 3450127 Solution, 1 L PC or Mac, includes darkroom, camera, cables, Criterion XT Tris-Acetate Gels 1610400 Coomassie Brilliant Blue R-250, 10 g Image Lab software; samples trays (#1708271, 3–8% Resolving Gel 3450129 3450130 3450131 — — 1610406 Coomassie Brilliant Blue G-250, 10 g 1708272, 1708273, or 1708274) are sold separately; Criterion Tris-HCl Gels sample trays are required to use the system 1610443 S ilver Stain Kit, includes oxidizer concentrate, silver 10% Resolving Gel 3450009 3450010 3450011 — 3450101 ™ TABLE CONTENTS OF reagent concentrate, silver stain developer, stains 1708265 Ch emiDoc XRS+ System with Image Lab 12.5% Resolving Gel 3450014 3450015 3450016 — 3450102 20 full size or 48 mini gels Software, PC or Mac, includes darkroom, UV 15% Resolving Gel 3450019 3450020 3450021 — — transilluminator, epi-white illumination, camera, 1610449 Silver Stain Plus™ Kit, includes fixative enhancer 4–15% Linear Gradient 3450027 3450028 3450029 — 3450103 power supply, cables, Image Lab Software 4–20% Linear Gradient 3450032 3450033 3450034 — 3450104 concentrate, silver complex solution, reduction ™ 10–20% Linear Gradient 3450042 3450043 3450044 — 3450107 moderator solution, image development reagent, 1709400 Personal Molecular Imager (PMI) System, development accelerator reagent, stains 13 full size PC or Mac, 110/240 V, includes sample tray set Criterion Tris-Tricine Gels or 40 mini gels and USB2 cable 16.5% Tris-Tricine 3450063 3450064 3450065† — — 1610496 Oriole™ Fluorescent Gel Stain, 1x solution, 1 L Analysis Software Criterion IEF Gels ™ 1709690 Image Lab Software pH 3–10 3450071† 3450072† 3450073† — — 1610492 Flamingo Fluorescent Gel Stain, 10x solution, pH 5–8 — 3450076† — — — 500 ml * Multichannel pipet compatible. 1703125 SYPRO Ruby Protein Gel Stain, 1x solution, 1 L ** Includes reference well(s). 1610440 Z inc Stain and Destain Kit, includes 125 ml of 10x *** 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; zinc stain solution A, 125 ml of 10x zinc stain solution 5,922,185; 6,162,338; and 6,783,651 and corresponding foreign patents. B, 125 ml of 10x zinc destain solution † Please allow up to 2 weeks for delivery. 1610470 Copper Stain and Destain Kit, includes 125 ml of 10x copper stain, 125 ml of 10x copper destain solution High-Throughput Stainers 1653400 Dodeca™ Stainer, large, 100–240 V, includes 13 trays (12 clear, 1 white), 12 tray attachments, shaking rack, solution tank, lid with shaker motor, shaker control unit, gel clip 1653401 Dodeca Stainer, small, 100–240 V, includes 13 trays (12 clear, 1 white), 12 Criterion tray attachments, shaking rack, solution tank, lid with shaker motor, shaker control unit, gel clip

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7 495 721 14 047 495 14 721 (0) 91 32 12700 Switzerland 52 555 488 The 7670 45 44 00 52 10 Finland 46 08 555 36 1 459 India 6100 Russia 7700 472 21 Russia 351

Mexico 4460

43 Belgium 89 1 877 177 01 3473 2 2 82

34 590 91 5200 Sweden Korea

48 22 331 99 99 Portugal 7000 7000

61 2 9914 2800 2 9914 61 Austria 6361

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27 (0) 27 861 246 723 Spain 47 23 38 41 30 23 Poland 3847 41 49 89 884 31 0 Hong Kong 852 2789 3300 Hungary United Kingdom 44 Kingdom United 4 8187300 020 971 8328 Emirates Arab United 200066 8311 2 651 216091

420 241 43086 8500 6169 420 21 241 532 Republic Czech Denmark 02

39 1 800 6723 424 Australia

Thailand Thailand Italy

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

963

South Africa South 3188 65 6415 1 905 364 3435 China 886 7189 2 2578 33 01 47 9533 47 69 01 65 Germany 972 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 and XT MES buffer kit, buffer XT MOPS XT MES running buffer, XT MOPS running buffer, gels, 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 for compensation by an or clinical analysis, in any event in return clinical information services, or providing other testing, analysis, or screening party. unrelated use only by the buyer of the product. Corporation, Carlsbad, CA, for Life Technologies under license from sold are standards Plus Protein Precision or its components. this product to sell or resell The buyer is not authorized für Bioanalytik GmbH to sell Bio-Rad Laboratories, Inc. is licensed by Institut by German patent application P 19641876.3. is covered StrepTactin use only. for research these products Number use only under U.S. Patent for research to sell SYPRO products Corporation is licensed by Bio-Rad Laboratories, Inc. 5,616,502. Corporation. Invitrogen trademarks of and SYPRO are NuPage, Pro-Q, Mac is a trademark of Apple Inc. Cy is a trademark of GE Healthcare. is a trademark of Dow Chemical für Bioanalytik GmbH. Triton Inc. Strep-tag is a trademark of Institut Probes, SYBR is a trademark of Molecular Company. is a trademark of Norton Corporation. Tygon Singapore Taiwan Taiwan Israel 2280Norway 64 9 415 Zealand New Web site USA bio-rad.com France Canada Canada Bio-Rad Laboratories, Inc.

Bulletin 6040 Ver C US/EG Life Science Group

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