Applications Tips and Techniques New Products Imaging BioRadiations AResource for Life Science Research Number 112

Cover Story Separations

Features Vivid Detail Detection Methods With 3.2 million pixels, the new VersaDoc™ Model 4000 system offers clear advantages in high-resolution imaging Drug Resistance Genotyping by FRET The differences are striking. The multi-megapixel resolution of the VersaDoc Model 4000 enables you to visualize samples that most imagers Methods for Purification cannot resolve. Multiple illumination and detection modes provide exceptional of His-Tagged application versatility. You can also combine pixels for greater sensitivity and optimize lens and illumination characteristics for image uniformity and accurate quantitation. All this, along with outstanding ease of use and a clear upgrade path, make the VersaDoc Model 4000 a definite standout. What’s New

Multi-Megapixel Resolution A Wide Range of Applications Chromatography Products in • 3.2 million pixels, with •Proteomics and genomics microlens technology • 1-D nucleic acid and protein gels Convenient Sizes Illumination Modes • 2-D protein gels MyCycler™ Sample Loading Tray •Trans- and epi-UV • Northern, Southern, and western blots • Dot and slot blots •Trans- and epi-white light ™ • Colony counting VersArray Hybridization Chamber Versatile Detection •Arrays • Fluorescent and More… • Colorimetric • Chemiluminescent • Chemifluorescent

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Bio-Rad Subsidiary to our readers Telephone Numbers Australia 61-2-9914-2800 Protein separation technologies, a traditional foundation for research, continue to find ever more Austria 43-1-877-89-01 applications. The shift from pure genomics toward an increased emphasis on understanding the roles of proteins is creating more demand for researchers familiar with these technologies. Belgium 32-9-385-55-11 Brazil 55-21-2527-3454 The authors of the cover story in this issue have broad experience in applications of protein Canada 00-1-905-712-2771 separations, from analytical sample preparation to large-scale production of pharmaceuticals. They China 86-21-6305-2255 draw on their personal expertise and Bio-Rad’s history of contributions to protein separation Czech Republic 420-2-41-43-05-32 technologies to illuminate why effective protein separation is indispensable to so many fields, Denmark 45-44-52-10-00 particularly proteomics and drug discovery. Finland 358-9-804-22-00 France 33-1-47-95-69-65 cover story Germany 49-89-318-84-0 Hong Kong 852-2-789-3300 12 Protein Separations: Proteomics to Production Hungary 36-1-455-8800 G Armin, W Gette, and U Snow, Bio-Rad Laboratories, Inc., Hercules, CA USA India 91-124-239-8112 Israel 972-3-951-4127 departments Italy 39-2-21609-1 2 What’s New Japan 81-3-5811-6270 Korea 82-2-3473-4460 21 Tips and Techniques Latin America 00-1-305-894-5950 24 New Literature Mexico 55-52-00-05-20 The Netherlands 31-318-540666 technical reports New Zealand 64-9-415-2280 6 ™ One Step Ahead Norway 47-23-38-41-30 Rapid Method Development With the BioLogic DuoFlow Chromatography Poland 48-22-331-99-99 System for the Purification of His-Tagged Proteins ™ F-F Wu, X He, and DR Nau, Bio-Rad Laboratories, Inc., Hercules, CA USA Introducing two new iScript one-step quantitative RT-PCR kits from Bio-Rad. Portugal 351-21-472-7700 Two kits completely in sync with your research — one step does it all Russia 7-095-721-1404 8 Drug Resistance Genotyping in Malaria Using FRET/Melt-Curve Analysis Singapore 65-6415-3188 S Decuypere, E Elinck, C Van Overmeir, U D’Alessandro, and JC Dujardin, Prince Leopold South Africa 27-11-4428508 Institute of Tropical Medicine, Antwerpen, Belgium ■ Versatile — kits for use with either SYBR® Green or Spain 34-91-590-5200 probe-based detection chemistries Sweden 46-8-555-12700 product focus article

■ Switzerland Sensitive — powerful combination of enzymes that 41-61-717-9555 10 The Bio-Rad Protein Family accurately detect 100 fg of input RNA Taiwan 88-62-2578-7189 Thailand 662-651-8311 ■ dimensions Convenient — one-tube reaction setup minimizes United Kingdom 44-20-8328-2000 handling and contamination risk USA Toll free 1-800-4BIORAD 18 Western Blot Detection Methods (1-800-424-6723) For more information, visit www.bio-rad.com/iscript/ On the cover: Legal Notices Conceptual illustration by Audra Geras. 8-Pette and 12-Pette are trademarks of Corning, Inc. ABI PRISM is a trademark of Applied Biosystems. Coomassie is a trademark of BASF Aktiengesellschaft. Costar is a trademark of Corning Costar Corporation. Lumigen is a trademark of Lumigen, Inc. Mac is a trademark of Apple Computer. SYBR, SYPRO, and Texas Red are trademarks of Molecular Probes. Bio-Rad Laboratories, Inc. is Applications Tips and Techniques New Products licensed by Molecular Probes, Inc. to sell SYPRO products for research use only, under US patent 5,616,502. Bio-Rad is licensed BioRadiations by Molecular Probes to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only. Tween is a AResource for Life Science Research Number 112 trademark of ICI Americas Inc. Windows and Windows 2000, NT, and XP are trademarks of Microsoft Corp. The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche, exclusively licensed to Cover Story Protein ™ ™ Separations Applied Biosystems. Use of the PCR process requires a license. The iCycler and MyCycler thermal cyclers are licensed for the

Features Western Blot PCR process. Additional licenses may be required from Applied Biosystems. Some applications may also require licenses from Detection Methods Drug Resistance Genotyping by FRET Methods for Purification other third parties. SYBR is a trademark of Molecular Probes. Bio-Rad is licensed by Molecular Probes to sell of His-Tagged Proteins

What’s New reagents containing SYBR Green I for use in real-time PCR, for research purposes only. Chromatography Products in Convenient Sizes MyCycler™ Sample Loading Tray VersArray™ Hybridization Chamber and More…

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Media Sampler Pack Updated iCycler iQ® Optical System Software Perfecting purification can require more than one choice of support. Bio-Rad now offers its most popular Obtain the most streamlined data output using the new version 3.1 software for the iCycler iQ optical system. This software update, media in a convenient sampler pack. The media sampler pack allows testing of different techniques at early available to all iCycler iQ customers, puts more analysis tools in the hands of the researcher. The iCycler iQ system software stages of process development. accelerates data analysis and optimizes graphical data output for publication needs. Features of the version 3.1 update include: Included in the sampler pack are: • Optimized curve-fit algorithm • Macro-Prep® High Q, DEAE, High S, • One-click toggle between logarithmic and linear views of data and CM ion exchange media • Rigorous autobaselining function • CHT™ ceramic hydroxyapatite Types I and II • UNOsphere™ Q and S ion exchange media Minimum computer specifications: • Macro-Prep methyl and t-butyl hydrophobic Windows NT, Windows 2000, or Windows XP operating system interaction media 500 MHz processor 256 MB RAM, 512 MB recommended All media are fully supported for regulatory 1024 x 768 screen resolution with true-color mode (24 or 32 bits) submissions. Microsoft Internet Explorer (v. 5.0 or higher) browser 6 GB hard drive Ordering Information CD-ROM drive Catalog # Description 57.6 kbps serial port 158-0100 Media Sampler Pack Bidirectional parallel port (EPP)

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Ordering Information ® Catalog # Description New Econo-Pac Cartridges 170-8754 iCycler iQ Optical System Software Version 3.1 Bio-Rad’s convenient and affordable prepacked Econo-Pac cartridges now include the popular UNOsphere™ S, UNOsphere Q, and Macro-Prep® DEAE ion exchange media, and CHT™ ceramic hydroxyapatite Type I. Econo-Pac cartridges simplify chromatography because there are no media to prepare or columns to pack. The cartridges are designed for use with the BioLogic™ systems, a peristaltic pump, any chromatography system, or a syringe for simple step elution. MyCycler™ Sample Loading Tray

Cartridge Name Support Type Chemical Form Protein Capacity Application The MyCycler sample loading tray is the newest accessory for the MyCycler personal + thermal cycler. The 96-well tray is compatible with individual tubes and is designed to UNOsphere Q UNOsphere Q Strong anion –N (CH3)3 900 mg BSA Protein, plasmid exchange purification simplify working with thin-wall PCR tubes. It can function as a tube rack during assay – UNOsphere S UNOsphere S Strong cation –SO3 300 mg BSA setup, support tubes during thermal cycling, and provide stackable storage for the tubes. exchange + The tray is placed directly over the MyCycler reaction block for easy sample loading DEAE Macro-Prep DEAE Weak anion –N (C2H5)2 175 mg BSA Acidic and neutral exchange protein and and removal, and is highly recommended for use with 0.2 ml thin-wall tubes to ensure peptide purification uniform contact between the heated lid of the MyCycler and each sample.

CHT I CHT ceramic Ceramic [Ca5(PO4)3OH]2 70 mg BSA Protein, nucleic acid hydroxyapatite hydroxyapatite purification Type I, 20 µm To request a MyCycler sample loading tray, contact Technical Support at 1-800-4BIORAD. To view Bio-Rad’s complete line of thermal cyclers and accessories, visit us on the Web at www.bio-rad.com/amplification/ Ordering Information Catalog # Description 1.33 cm Ordering Information Econo-Pac Cartridges Catalog # Description 732-0200 UNOsphere Q Support, 1 x 5 ml 3.6 cm 170-9709 MyCycler Sample Loading Tray 732-0201 UNOsphere Q Support, 5 x 1 ml 732-0202 UNOsphere Q Support, 5 x 5 ml 732-0210 UNOsphere S Support, 1 x 5 ml 732-0211 UNOsphere S Support, 5 x 1 ml 0.59 cm 732-0212 UNOsphere S Support, 5 x 5 ml 732-0007 Macro-Prep DEAE Support, 1 x 5 ml 3.6 cm 732-0008 Macro-Prep DEAE Support, 5 x 5 ml 732-0009 Macro-Prep DEAE Support, 5 x 1 ml 732-0082 CHT Ceramic Hydroxyapatite Type I Support, 1 x 5 ml 732-0086 CHT Ceramic Hydroxyapatite Type I Support, 5 x 1 ml 732-0084 CHT Ceramic Hydroxyapatite Type I Support, 5 x 5 ml

2 BioRadiations 112 BioRadiations 112 3 what’s new what’s new

™ ® Ordering Information ™ iTaq SYBR Green Supermix With ROX Catalog # Description VersArray Hybridization Chamber iTaq SYBR Green supermix with ROX is formulated to easily achieve optimal 1.0e+002 iTaq SYBR Green Supermix With ROX 170-8850 iTaq SYBR Green Supermix With ROX, The Ultimate Tool for Microarray Hybridizations results in real-time quantitative PCR assays on all ROX-dependent instrument 200 x 50 µl reactions platforms. This formula delivers high performance with cDNA as well as 1.0e+001 170-8851 iTaq SYBR Green Supermix With ROX, The VersArray hybridization chamber is designed to increase the effectiveness of genomic and plasmid DNA templates over a broad dynamic range, achieving 500 x 50 µl reactions manual (coverslip) hybridizations. It allows the quick and easy loading of one or sensitive and specific amplification over at least 7 orders of magnitude. The 170-8852 iTaq SYBR Green Supermix With ROX, two slides, permits superior thermal transfer, and is watertight to allow

Delta Rn 1,000 x 50 µl reactions supermix is preblended with hot-start iTaq DNA polymerase, optimized buffer, 1.0e+000 hybridization in a water bath. The chamber is easily opened and closed using the nucleotides including dUTP for optional UNG-mediated treatment of single attachment bolt, and a unique patent-pending pressure mechanism ensures carryover contamination, SYBR Green I dye, and ROX passive reference dye. uniform sealing. 1.0e-001 Its convenient one-tube formulation will enable you to obtain specific and 246 81012 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 sensitive amplification every time. Cycle Number Key Benefits

Bio-Rad’s supermix gives superior results on the ABI PRISM • Simultaneous processing of two microarray slides • Proprietary low-foam formulation enables smoother pipetting 7000 sequence detection system. iTaq SYBR Green supermix with • Watertight — allows water bath incubations • Validated for use on all ROX-dependent optical thermal cycler platforms ROX (green) and other ROX-containing supermixes (red and blue) • One attachment bolt for easy and convenient opening and closing • Conveniently preblended with ROX and dUTP were used to amplify 10-fold serial dilutions (107 to 10 copies) of the • Water reservoirs (2 per slide) prevent slides from drying out GAPDH gene in a plasmid template. The Bio-Rad supermix • Aluminum composition promotes uniform heat transfer consistently generated earlier CT values than the competitors’ mixes, ∆ For more information, request bulletin 3065. and produced high Rn values.

Water reservoirs prevent drying Ordering Information Catalog # Description ™ iScript One-Step Quantitative RT-PCR Kits iScript One-Step Quantitative RT-PCR Kits 170-8892 iScript™ One-Step RT-PCR Kit With SYBR® Green, 50 reactions The iScript one-step RT-PCR kits (with or without SYBR Green) are highly sensitive solutions for real-time quantitative RT-PCR on 170-8893 iScript One-Step RT-PCR Kit a broad range of optical thermal cyclers. cDNA synthesis and PCR amplification are conveniently carried out in the same tube, using With SYBR Green, 200 reactions the powerful iScript reverse transcriptase and iTaq™ hot-start DNA polymerase combination. These kits are optimized to deliver 170-8894 iScript One-Step RT-PCR Kit for Probes, 50 reactions maximum RT-PCR efficiency, sensitivity, and specificity without compromising fluorescent signal. The proprietary reaction buffers have 170-8895 iScript One-Step RT-PCR Kit for Probes, been formulated for optimal activity of both iScript reverse transcriptase and iTaq DNA polymerase, while minimizing the potential for 200 reactions formation of primer-dimers and other nonspecific PCR artifacts. The kits are easy to use and are perfectly suited for a broad range of Other PCR Reagents real-time PCR applications. 170-8890 iScript cDNA Synthesis Kit, 25 x 20 µl reactions 170-8891 iScript cDNA Synthesis Kit, • Highly specific amplification over a broad dynamic range 100 x 20 µl reactions • Extremely sensitive detection down to 100 fg of input RNA 170-8860 iQ™ Supermix, 100 x 50 µl reactions • Convenient one-tube setup minimizes handling and contamination risk 170-8862 iQ Supermix, 500 x 50 µl reactions 170-8864 iQ Supermix, 1,000 x 50 µl reactions 170-8880 iQ SYBR Green Supermix, For more information, request bulletin 3066. 100 x 50 µl reactions 170-8882 iQ SYBR Green Supermix, O-rings for a Central bolt “X” distributes Anodized 500 x 50 µl reactions 1,000 watertight seal pressure uniformly aluminum 10,000 170-8884 iQ SYBR Green Supermix, base 1,000 x 50 µl reactions 170-8870 iTaq DNA Polymerase, 250 U Ordering Information 170-8872 MgCl2 Solution, 50 mM, 1.25 ml 170-8874 dNTP Mix, 200 µl of 10 mM Catalog # Description of each dNTP 169-0500 VersArray Hybridization Chamber 100 100 PCR base line subtracted RFU PCR base line subtracted CF RFU 10 10 051015 20 25 30 35 40 45 051015 20 25 30 35 40 45 Cycle Cycle The iScript one-step RT-PCR kit with SYBR Green provides Bio-Rad’s iScript one-step RT-PCR kit for probes gives high sensitivity across a broad range of concentrations. accurate and streamlined results with any detection One-step RT-PCR reactions were performed in quadruplicate, chemistry. RNA (100 ng to 100 fg) isolated from HeLa cells using along with no-template controls, using GAPDH primers and 100 ng the Aurum total RNA kit was reverse transcribed and amplified to 100 fg of total HeLa RNA. Reactions were carried out on the using primers to β-actin and a FAM-labeled detection probe. The iCycler iQ® real-time system. Standard curve had r = 1.000, reactions were carried out on the MyiQ™ real-time system. Standard slope = –3.466, efficiency = 95%. curve had r = 1.000, slope = –3.360, efficiency = 98.4%.

4 BioRadiations 112 BioRadiations 112 5 technical report technical report

Methods 2.5 Imidazole Fig. 2. Effect of imidazole concentration Setup concentration on resolution 2.0 Unbound 75 mM The column, UV and conductivity detectors, fraction 100 mM of the protein complex. The collector, and three SV5-4/AVR7-3/AVR9-8 valves 250 mM 75 mM sample is aligned to were connected to the workstation and controller as 500 mM the y-axis; the traces for other 1.5 750 mM concentrations are offset to Rapid Method Development With the illustrated in Figure 1. All nine buffers, with the faciliate comparison. 280 Peak 1 ™ following compositions, were filtered and connected A Peak 2 BioLogic DuoFlow Chromatography System to the indicated SV5-4 and AVR9-8 valve ports: 1.0 • A1: discharging buffer (50 mM potassium for the Purification of His-Tagged Proteins phosphate, 50 mM EDTA, 300 mM NaCl, pH 7.5) 0.5 • A2: cleansing buffer (50 mM potassium acetate, Fang-Fang Wu, Xuemei He, and David R Nau, Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA 300 mM NaCl, pH 4.0) 0.0 • A3: charging buffer (100 mM NiSO , 300 mM 30 35 40 45 4 Run time, min Introduction and recovery requirements. Usually, these would NaCl, pH 4.3) Immobilized metal affinity chromatography require a series of tedious and repetitive operations • A4: loading buffer (50 mM potassium phosphate, Imidazole concentration (IMAC) is a powerful technique that can be used by the researcher, including the discharging and 300 mM NaCl, pH 8.0) 75 100 250 500 750 mM Fig. 3. Protein fractions from buffer scouting. for the efficient purification of recombinant His- recharging of the column with fresh metal ions after • B1: elution buffer 1 (loading buffer + 75 mM 12 3 4 5 6 7 8 910111213 tagged proteins from a variety of expression systems. every run in order to ensure reproducible results. imidazole) Fractions were run on a Tris-HCl 4–16% gel and The synthesis of IMAC resins begins with the Bio-Rad has developed an extremely rapid and • B2: elution buffer 2 (loading buffer + 100 mM stained with Coomassie Blue. derivatization of an appropriate wide-pore base resin efficient method using the BioLogic DuoFlow high- imidazole) Lanes 1 and 13, molecular with metal chelating groups, such as iminodiacetic resolution chromatography system to purify a 75 kD • B3: elution buffer 3 (loading buffer + 250 mM weight markers; lanes 3, 5, 7, 9, peak 1; lanes 2, 4, 6, 8, acid (IDA) or nitrilotriacetic acid (NTA). Specific His-tagged protein from crude E. coli lysate on a imidazole) 10, peak 2; lane 11, unbound 2+ 2+ 2+ 2+ ™ transition metal ions — usually Cu , Ni , Co , Ni -charged UNOsphere IMAC resin (soon to be • B6: elution buffer 4 (loading buffer + 500 mM protein; lane 12, crude E. coli Ca2+, or Zn2+ — are then immobilized to form a launched). In the present study, the concentration imidazole) lysate. Arrow indicates charged support. IMAC resins exhibit high affinity of a competitive eluting agent, imidazole, was • B7: elution buffer 5 (loading buffer + 750 mM location of 75 kD His-tagged protein. Peak 1 contained and metal-dependent selectivity for His-tagged optimized using the BioLogic DuoFlow system by imidazole) impurities + 75 kD His-tagged proteins, as well as naturally occurring proteins that executing multiple repetitive chromatography runs protein. Peak 2 contained the are rich in histidine, cysteine, aspartic acid, or with a nine-buffer system. The plumbing setup of Experimental Results 75 kD His-tagged protein. glutamic acid residues. the system included a 5-port/4-way SV5-4 valve A 1 ml column (0.5 cm ID x 5 cm) was packed The elution profile during each run was monitored To obtain highly purified proteins from an IMAC and a 9-port/8-way AVR9-8 valve, which were both manually with Ni2+-charged UNOsphere IMAC at 280 nm, and a series of 1 ml fractions was collected system, a series of method development designed for flexible automated operation. This resin (60 µm). Five separate runs were conducted, using a BioFrac™ fraction collector. is typically required. These experiments involve multiple inlet port configuration, coupled to the with either 75, 100, 250, 500, or 750 mM imidazole The resulting chromatograms obtained with the optimization of the mobile phase pH, buffer system, buffer scouting feature of the BioLogic DuoFlow in the elution buffer. Following elution and a 75, 100, 250, 500, and 750 mM imidazole elution flow rate, concentration of the competitive eluting software, allowed us to complete this subsequent wash step, the column was regenerated buffers were superimposed using the Trace Compare agents, and a number of other potential parameters with a single linked scouting run and with virtually using the discharging and charging buffers listed feature provided within the BioLogic DuoFlow that may also be examined, depending on purity no user intervention. under Setup. These five successive runs were linked software; these are illustrated in Figure 2. through the buffer scouting function of the BioLogic The data in Figures 2 and 3 indicate that optimal DuoFlow software. For each run, the column was results were obtained with an elution buffer preequilibrated with 10 column volumes (CV) of consisting of 250 mM imidazole, 50 mM potassium AVR7-3 injection loading buffer at a flow rate of 1.5 ml/min. Crude phosphate, 300 mM NaCl, pH 8.0. This elution Fig. 1. System 6 5 valve plumbing diagram. 7 E. coli lysate (250 µl) containing a 75 kD His-tagged buffer provided the most effective separation and protein was loaded into a 3 ml injection loop and removal of the impurities (Figure 3, lanes 5 and 6, Load 4 Sample injected into the column through an AVR7-3 valve. respectively). Mixer 1 loop Column Host protein contaminants (which were not 3 2 retained by the IMAC resin) were removed by Conclusions

BioLogic DuoFlow washing the column with 10 CV of the loading The development of rapid and efficient methods for workstation Sample buffer. The 75 kD His-tagged protein was then the chromatographic isolation and characterization inject UV eluted with 5 CV of imidazole elution buffer, of biomolecules requires a chromatography system detector followed by an additional wash with 10 CV of the that is capable of advanced automated operation AB loading buffer to eliminate residual proteins from the with minimal user intervention. This study Conductivity column. The column was then regenerated as demonstrates that the sophisticated valve con- monitor detailed in the following steps using 10 CV of each figuration, high degree of flexibility, and versatile 4 5 buffer: metal removal with the discharging buffer; software of the Biologic DuoFlow system make it 2 1 3 6 washing with the cleansing buffer; recharging of the ideal for meeting the demands of a modern method metal ion with the charging buffer; removal of excess development laboratory. 7 4 3 2 metal ions with the cleansing buffer; and finally, reequilibration with the loading buffer. For more information on Bio-Rad’s entire BioLogic A1 A2 1 8 B3 B6 In order to confirm the separation of the target chromatographic system, request bulletin 2687A. A4 protein from other E. coli proteins in the crude sample, A3 B2 B1 B7 the peak fractions were analyzed by electrophoresis on SV5-4 valve AVR9-8 valve BioFrac™ fraction collector a Tris-HCl 4–16% gradient polyacrylamide gel.

6 BioRadiations 112 BioRadiations 112 7 technical report technical report

51T 59T PCR product will have an intermediate 5 T . Analysis of the melt curve allows determination 4 Fig. 2. First negative m derivative of melt curves of the DHFR genotype of codons 51 and 59 of the 3 for DHFR clones. Plasmids parasite population present in the sample. 2 analyzed were 51T 59C DHFR (FR-V1S, red), 51T 59T –dF/dT Methods 1 DHFR (N5, green), and 51A Drug Resistance Genotyping in Malaria Using 59T DHFR (FR-3D7, blue); 0 Design of FRET Primer and Hybridization Probe also shown is no-template FRET/Melt-Curve Analysis The forward primer was labeled 6 nucleotides from –1 control (gray). Melting peaks 46 48 50 52 54 56 58 60 62 64 66 68 70 were identified at 53.7°C, the 3' end with ROX, in order not to obstruct Temperature, °C Saskia Decuypere, Ellen Elinck, Chantal Van Overmeir, Umberto D’Alessandro, and Jean-Claude Dujardin, Prince Leopold Institute of extension of the primer during PCR. 58.3°C, and 62.4°C, Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium; Correspondence address: [email protected] respectively. Horizontal blue The probe was specifically designed to achieve FR-50 has a DHFR insert that also contains the line indicates threshold for background fluorescence. a maximum difference in Tm between the different mutated codon 51, and a point mutation in codon 50 Introduction and 59 (T→C) increase resistance to the drug genotypes as calculated with Meltcalc software at the penultimate position of the 3' end of the probe,

Sulfadoxine-pyrimethamine (SP) is an antifolate (Peterson et al. 1988). Molecular tools for (developed by E Schütz and N von Ahsen; which has only a minor influence on the Tm of the drug for the treatment of malaria. Pyrimethamine genotyping such mutations could be used for the downloadable at http://meltcalc.com) (Schütz and probe. The melt curves of the mixtures (Figure 3) binds and inhibits Plasmodium falciparum surveillance of SP resistance in countries where von Ahsen 1999). The probe complements the wild- contained the two expected peaks that correspond to dihydrofolate reductase (DHFR), a key enzyme for malaria is endemic. type antisense strand of the PCR product and was the two different genotypes. The percentage of each DNA synthesis. However, the widespread use of SP In this report, we present a new assay based on labeled at the 3' end with FAM. Primers and probe genotype in the mixtures roughly concurred with the has led to a rapid emergence of SP-resistant fluorescence resonance energy transfer (FRET) were synthesized by Eurogentec. area underneath the peak that represents that parasites. Mutation of the DHFR gene at codon 108 during melt-curve analysis that is able to determine genotype, as expected. (A→T) causes resistance to pyrimethamine. the genotype of parasites at codons 51 and 59 of the PCR and Melt-Curve Analysis Conditions 3 Additional point mutations at codons 51 (A→T) DHFR gene. The FRET assay design involves (1) a The target strand to which the FAM-labeled probe A Fig. 3. First negative 2 probe specific for the DHFR gene that encompasses binds was produced in excess using an asymmetric derivative of melt curves Step 1. DHFR PCR with ROX primer codons 51 and 59 labeled with FAM at the 3' end, PCR. Amplification reactions (50 µl) contained 1 of experimental mixtures.

Fig. 1. Schematic ROX forward primer and (2) a primer for amplification of the DHFR 1x iQ™ supermix (Bio-Rad), 500 nM forward primer, –dF/dT Red curves: A, 50% FR-V1S representation of the 0 and 50% FR-50; B, 90% FR- gene placed adjacent to the probe and labeled with and 100 nM reverse primer. The probe was added FRET/melt-curve –1 V1S and 10% FR-50. Gray assay. The assay was ROX ROX near its 3' end. Consequently, upon hybrid- immediately after amplification at a final curve represents no-template 3 designed to genotype 5' 3' ization of the probe to the PCR product and excitation, concentration of 160 nM. B control; horizontal blue line codons 51 and 59 of 2 indicates threshold for Plasmodium falciparum 3' 5' FRET can occur from FAM to ROX (Figure 1). The DNA template used in these experiments background fluorescence. DHFR. The assay basically consists of two steps (Figure consisted of DHFR clones with a Plasmodium DHFR 1

Step 2. Addition of FAM probe and melt-curve analysis –dF/dT • = codons 51 and 59 of 1). The first step is a standard PCR using the ROX- insert with known genotype at codons 51 and 59. 0 ® Plasmodium falciparum ROX labeled forward primer and a reverse primer that Bio-Rad’s iCycler iQ system was used to perform 5' 3' –1 DHFR; X = 490/20X FAM amplifies a fragment of the Plasmodium DHFR gene. the PCR and melt-curve analysis. PCR conditions 46 48 50 52 54 56 58 60 62 64 66 68 70 excitation filter; M = 620/ During this PCR, the product is labeled with ROX were: initial denaturation at 94°C for 5 min, Temperature, °C 30M ROX emission filter. 3' FAM At 94°C as the forward primer is extended. Following followed by 35 cycles at 95°C for 30 sec, 52°C for Discussion amplification, the FAM-labeled probe is added to 1 min, 72°C for 1 min, and a final extension at 72°C Melt-curve analysis using FRET technology is rapidly the obtained PCR products. The resulting mixture is for 8 min. The melt-curve protocol that followed emerging as an efficient genotyping method. However, 5' FAM probe denatured and then cooled to 10°C below the addition of the probe consisted of two steps: (1) most FRET genotyping assays can only detect one

melting temperature (Tm) of the probe, allowing the 1 min at 94°C, (2) 110 repeats of heating for 30 sec, point mutation. The new DHFR genotyping assay T probe to anneal adjacent to the ROX fluorophore of starting at 48°C with 0.3°C increments. presented here clearly demonstrates that with a simple the PCR product. The temperature is subsequently All experiments were carried out in triplicate to FRET primer/probe design it is feasible to detect slowly increased, while ROX fluorescence resulting verify reproducibility. mutations at two (or more) nucleotides simultaneously. Fluorescence from FRET is continually monitored. When the Furthermore, we show that it is possible to quantitate detected Tm of the probe/PCR product hybrid is reached, Results the different genotypes present in a polyclonal parasite ROX M 5' 3' ROX fluorescence will decrease as FRET can no The plots of –dF/dT of the melt curves for three population, which is not possible with any other 3' 5' longer occur between the FAM label of the probe different clones containing respectively 51A 59T molecular tool. Consequently, FRET/melt-curve FAM X and the ROX label of the PCR product. DHFR (plasmid FR-3D7), 51T 59T DHFR (clone analysis promises to be a very useful genotyping tool At 48°C The change in ROX fluorescence appears as a N5), and 51T 59C DHFR (plasmid FR-V1S) are for all infectious diseases. FRET positive peak on a plot of the –dF/dT (first negative shown in Figure 2. The three different genotypes Light source derivative of the fluorescence with respect to temp- had distinct melting peaks, with 51A 59T at 62.4°C, References erature function) vs. temperature. However, the 51T 59T at 58.3°C, and 51T 59C at 53.7°C. Peterson DS et al., Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in T melting temperature of the probe/PCR product Since a Plasmodium population in a sample might Falciparum malaria, Proc Natl Acad Sci USA 85, 9114–9118 (1988) hybrid will depend on the nucleotide sequence of be polyclonal (that is, some parasites have 51T 59T Schütz E and von Ahsen N, Spreadsheet software for thermodynamic No fluorescence codons 51 and 59. The probe was designed as an and some have 51T 59C), this assay should also be melting point prediction of oligonucleotide hybridization with and detected exact complement of the 51A 59T (wild-type) capable of identifying and quantitating multiple without mismatches, Biotechniques 27, 1218–1224 (1999) ROX M 5' 3' sequence. If a mutation is present at codon 51, 59, or DHFR genotypes in one sample. Therefore, Figures 2 and 3, © 2003 by the Infectious Diseases Society of

5' both in the PCR product, the mismatches make the experimental mixtures containing different plasmids America. Reproduced from Decuypere S et al., J Infect Dis 188, FRET X X hybrids weaker, resulting in a lower melting temp- were also assayed. The following mixtures were tested: 1245–1249 (2003) with permission from the University of At T of hybrid m erature compared to the hybrid with no mismatches. (1) 50% FR-V1S/50% FR-50 and (2) 90% FR-V1S/ Chicago Press. Thus, a 51A 59T PCR product will have a much 10% FR-50. Plasmid FR-V1S contains a DHFR gene 3' Light source M higher Tm than a 51T 59C PCR product, while a with a point mutation in codons 51 and 59. Plasmid FA

8 BioRadiations 112 BioRadiations 112 9 product focus product focus

References Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248–254 (1976) Lowry OH et al., Protein measurement with the Folin phenol reagent, J Biol Chem 193, 265–275 (1951) For more information on Bio-Rad’s protein assay family, request bulletins 2969 and 1069. For more The Bio-Rad Protein Assay Family information on microplate systems, request bulletins 2727, 2733, 2246, 2581, and 2054. For more information on the SmartSpec Plus spectrophotometer, request bulletins 2826 and 2827. Determining the concentration of protein samples is critical for many experiments. In a typical colorimetric protein assay, a chemical reagent is added to a protein sample solution, producing a color change that is Ordering Information measured with a spectrophotometer or microplate reader and compared to a standard curve of known Catalog # Description protein concentrations. Quick Start Bradford Protein Assay 500-0201 Quick Start Bradford Protein Assay Kit 1, includes 1 L 1x dye reagent, When you need to determine the concentration of protein in your sample, choose one of Bio-Rad’s protein bovine serum albumin standard (5 x 2 mg/ml) assays. No matter what chemical components are contained in the buffer or the amount of protein available 500-0202 Quick Start Bradford Protein Assay Kit 2, includes 1 L 1x dye reagent, for assay in your sample, Bio-Rad has an assay solution for you. Bio-Rad offers four protein assays, each with a bovine serum albumin standard set (2 sets of 7 standards, 0.125–2.0 mg/ml) unique set of advantages. They are compared in the table below. 500-0203 Quick Start Bradford Protein Assay Kit 3, includes 1 L 1x dye reagent, bovine γ-globulin standard, (5 x 2 mg/ml) 500-0204 Quick Start Bradford Protein Assay Kit 4, includes 1 L 1x dye reagent, new bovine γ-globulin standard set (2 sets of 7 standards, 0.125-2.0 mg/ml) ™ Quick Start Bio-Rad 500-0205 Quick Start Bradford 1x Dye Reagent, 1 L Bradford (Bradford) DC™ Protein RC DC™ Protein 500-0206 Quick Start Bovine Serum Albumin Standard, 5 x 2 ml vials of 2 mg/ml Protein Assay Protein Assay Assay Assay 500-0207 Quick Start Bovine Serum Albumin Standard Set, 2 sets of 7 standards, Type of protein assay Ready to use, Reducing agent Detergent Reducing agent reducing agent compatible compatible and detergent 0.125–2.0 mg/ml compatible compatible 500-0208 Quick Start Bovine γ-Globulin Standard, 5 x 2 ml vials of 2 mg/ml γ Standard assay sample volume 100 ml 100 ml 100 ml 100 ml 500-0209 Quick Start Bovine -Globulin Standard Set, 2 sets of 7 standards, 0.125–2.0 mg/ml Microplate assay sample volume 5 ml 10 ml 5 ml — Bio-Rad (Bradford) Protein Assay Linear range for standard assay 0.125–1.5 mg/ml 0.2–1.5 mg/ml 0.2–1.5 mg/ml 0.2–1.5 mg/ml 500-0006 Bio-Rad Protein Assay Dye Reagent Concentrate, 5x, based on method of Minimum incubation time 5 min 5 min 15 min 15 min Bradford, 450 ml Dilution of dye reagent No Yes Yes Yes 500-0001 Bio-Rad Protein Assay Kit I, includes 450 ml dye reagent concentrate, and standards required bovine γ-globulin standard Prediluted standard set Yes No No No 500-0002 Bio-Rad Protein Assay Kit II, includes 450 ml dye reagent concentrate, bovine wavelength 595 nm 595 nm 750 nm 750 nm serum albumin standard Adapted from method of Bradford (1976) Bradford (1976) Lowry et al. (1951) Lowry et al. (1951) DC Protein Assay 500-0111 DC Protein Assay Kit I, includes 250 ml alkaline copper tartrate solution, 2 L dilute Folin reagent, 5 ml surfactant solution, bovine γ-globulin standard Instruments for Quantitation 500-0112 DC Protein Assay Kit II, includes 250 ml alkaline copper tartrate solution, Pair the protein assay kits with Bio-Rad’s spectrophotometers and microplate readers for great results. 2 L dilute Folin reagent, 5 ml surfactant solution, bovine serum albumin standard ™ 500-0113 DC Protein Assay Reagent A, 250 ml alkaline copper tartrate solution The SmartSpec Plus spectrophotometer is ideal for routine quantitation of protein samples. This simple- 500-0114 DC Protein Assay Reagent B, 1 L dilute Folin reagent to-use instrument has preprogrammed methods that complement all of Bio-Rad’s protein assay kits. The 500-0115 DC Protein Assay Reagent S, 5 ml surfactant solution SmartSpec Plus automatically calculates standard curves and determines the concentration of unknown 500-0116 DC Protein Assay Reagents Package, includes 250 ml alkaline copper tartrate samples. Also available are a line of high-quality protein assay cuvettes that are compatible with both the solution, 2 L dilute Folin reagent, 5 ml surfactant solution protein assay kits and the SmartSpec Plus. RC DC Protein Assay 500-0121 RC DC Protein Assay Kit I, includes RC reagents package, DC protein assay reagents package, bovine γ-globulin standard; 500 standard assays Microplate Systems 500-0122 RC DC Protein Assay Kit II, includes RC reagents package, DC protein assay Simplify your assay procedures using 6- to 1,536-well microplates and Bio-Rad’s extensive selection of reagents package, bovine serum albumin standard; 500 standard assays microplate absorbance readers. State-of-the-art software provides features for high throughput, sophisticated 500-0120 RC DC Protein Assay Reagents Package, includes RC reagents package, DC protein assay reagents package; 500 standard assays curve fit analysis, and complex kinetic analysis. 500-0119 RC Reagents Package, contains RC reagent I (250 ml), RC reagent II (250 ml); 500 standard assays 500-0117 RC Reagent I, 250 ml 500-0118 RC Reagent II, 250 ml Protein Standards 500-0005 Protein Standard I, bovine γ-globulin, reconstituted volume 20 ml 500-0007 Protein Standard II, bovine serum albumin, reconstituted volume 20 ml Related Products 223-9950 Standard Disposable Polystyrene Cuvettes, 3.5 ml, 100 Quick Start Bradford Bio-Rad (Bradford) DC Protein Assay RC DC Protein Assay Disposable Cuvettes 223-9955 Semimicrovolume Disposable Polystyrene Cuvettes, 1.5 ml, 100 Protein Assay Protein Assay 224-0096 Costar 96-Well Flat-Bottom EIA Plates, polystyrene, 5 per package, box of 100 224-4888 8-Pette Adjustable-Volume 8-Channel Pipet, 20–200 µl 224-4880 12-Pette Adjustable-Volume 12-Channel Pipet, 20–200 µl 170-2525 SmartSpec Plus Spectrophotometer 170-6930 Benchmark Plus Microplate Spectrophotometer, PC, with temperature control 168-1000 Model 680 Microplate Reader 170-9500 Ultramark Microplate System 170-7009 Model 1575 Immunowash Microplate Washer

SmartSpec Plus Benchmark™ Plus Microplate Model 680 Microplate Ultramark™ Microplate Model 1575 Immunowash Spectrophotometer Spectrophotometer Reader Systems Microplate Washer

10 BioRadiations 112 BioRadiations 112 11 cover story cover story Protein Separations Proteomics to Production

Investigations of many of the most intriguing electrophoresis and chromatography. Both exploit biological questions today are dependent on a better inherent protein characteristics. Electrophoresis — understanding of individual proteins. With the the application of an electric current to a solid-phase human genome now unraveled, the complementary support such as a gel in which proteins can migrate protein products’ roles, most of which remain to be — can separate proteins based on their isoelectric discovered, have become a major focus. Endeavors point, size (molecular weight), or both (Figure 1). to map out the proteins associated with their gene Chromatography — the differential partitioning of counterparts are critical to understanding questions proteins between stationary and mobile phases — such as the profiles of diseases and how to control separates proteins based on their charge, size, them. Many different approaches are directed at hydrophobicity, or affinity for particular these efforts, with protein separation remaining of compounds (Figure 2). Although both techniques paramount importance. have been in common use for some time, they Protein separation is used to fractionate a continue to be used in new scientific fields. complex sample so it can be subjected to further analysis or experimentation. With pure fractions, Why Separate Proteins? researchers are able to better understand the Protein separations are invaluable to proteomics, function of a particular protein in a biological drug discovery, and production. In both areas, steps system. Two distinct but complementary include preliminary sample preparation, protein technologies are used to separate most proteins: identification and characterization, and purification at both laboratory and process scales. A Proteomics, or the study of the function of all Fig. 1. Electrophoresis. + – expressed proteins, is heavily dependent on protein In 2-D SDS-PAGE, proteins 7.1 7.1 separation. Using the foundations of protein in a mixture are first applied 8.4 7.1 8.8 3.9 8.4 8.8 3.9 8.8 chemistry, proteomics has refined many commonly to an IPG strip (panel A). The 8.4 proteins are focused in the 3.7 5.3 3.7 5.3 used techniques such as two-dimensional (2-D) gel strip to their isoelectric points Before focusing electrophoresis. Often called the “workhorse of (B). The strip is then applied proteomics”, 2-D separates proteins to a slab gel containing SDS, B in which they are separated pH 3 10 in two dimensions, the first by isoelectric point and by molecular weight (C). the second by size (Figure 1). 2-D separations of 7.1 3.93.9 7.1 8.48.4 8.88.8 3.73.7 5.35.3 7.1 8.4 8.8 complex protein samples reveal less-abundant and often overlooked proteins.

After focusing Drug discovery is another major driver in the separation of proteins. Pharmaceutical and biotechnology companies search for new proteins C and other compounds to cure diseases, utilizing both electrophoresis and chromatography as tools. 3.9 8.8 MW Screening assays are employed to determine the biological effect of a protein target. In order to perform these assays, researchers must first separate and purify proteins of interest. Chromatographic 7.1 8.4 methods isolate and purify target proteins from other sample material for further screening and 3.7 5.3 identification. These methods allow flexibility in choice of mobile phase and support, in addition to

SDS-charged proteins resolved according to size in SDS-PAGE gel scalability. After proteins of interest are identified, Illustration by Audra Geras by Gabriella Armin, William Gette, and Ursula Snow, Bio-Rad Laboratories, Inc., Hercules, CA USA

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specific binding to an antibody or other ligand, and match to a database entry, knowledge of the purity, and chromatography is the most often used Fig. 2. Chromatography. A solution containing a biological activity or other screening assays. More sequence and inferred structure of the protein can technique to achieve this goal. mixture of proteins is typically definitive characterization includes peptide mass or still provide clues to its function and possible Determination of posttranslational modifications applied to a column sequence determination, X-ray crystallography to interactions with other proteins, based on its other is often required for the growing field of cell containing a stationary determine 3-D structure, evaluation of similarities to known proteins. regulation studies. Such modifications can be support. Proteins in the mixture interact differently posttranslational modifications such as glycosylation X-ray crystallography is the definitive method characterized using antibodies, by electrophoresis, with the support, leading to and phosphorylation, and analyses of the for determining protein structure. Knowledge of a often combined with modification-specific stains separation. The characteristics biochemical and cellular functions of the protein. protein’s molecular structure allows a better (for example, to detect phosphorylation), and by of the support and the solutions used to wash and Each of these characterizations helps to positively understanding of how it works and can lead to mass spectrometric peptide analysis. elute the proteins can be identify the protein in later studies, and most rely to better drugs and treatments for disease. Effective More complete characterization would include modified to optimize some extent on the science of protein separation. crystallography experiments require preparation of biochemical definition of the role of the protein in separation. Molecular weight estimation by SDS-PAGE is highly pure protein. Significant effort and expense the cell. Definitive characterization may require routinely used for rapid preliminary identification is required to purify proteins to greater than 99% complex assays or other tools. large-scale purifications, done exclusively through of proteins, particularly analysis of chromatographic chromatographic means, are utilized. Analytical column fractions, based on comparison to migration Protein Separation Techniques: A Research Perspective assessment of purity is often checked through 1-D gel of suitable protein standards in the same gel. This electrophoresis, a separation technique used as a method may not be reliable for complex samples, complement to chromatography. which may contain several proteins of the same Using Proteomics to Enhance the Discovery of New Drugs molecular weight. In this case, 2-D electrophoretic Mark Molloy, Senior Scientist, Pfizer Global R&D Sample Preparation analysis, which separates on the basis of both Proteomics encompasses large-scale, high- Pharmaceutical companies use proteomics to gain insight The first step in any protein separation process is isoelectric point and size, is often used to identify throughput, parallel protein profiling. The into compound safety, mechanism of action, and for target sample preparation. Proteins are labile, so and quantitate proteins of interest. 2-D gel application of proteomics requires two distinct identification. The concept is to shorten the timeline from idea precautions must be taken to minimize protein electrophoresis also increases overall sample skill sets: analytical chemistry for protein to drug by applying molecular technologies such as proteomics degradation. Other obstacles in isolating and resolution, which ultimately leads to an increase separation, identification, and characterization, and expression profiling. An emerging focus is to use proteomics purifying proteins include limited amount of sample, in the number of protein identifications possible and biochemistry/physiology for interpreting the for biomarker discovery, again to shorten the timeline from new variable sample composition, the presence of within a sample. significance of the analytical measurements. drug to market. nonprotein contaminants, wide differences in A widely used method for specific protein I started my research examining the evolutionary relationship Improvements in speed, sensitivity, automation, and relative protein abundance within a sample, and identification is western blotting. Following 1-D gel between the kangaroo and platypus by studying their milk whey robustness are key areas for attention. From the viewpoint of the existence of isozymes and posttranslational electrophoresis, protein bands are transferred to a proteins. Two-dimensional electrophoresis was used for protein pharmaceutical applications, one major goal is an integrated purification and Edman degradation provided sequence analysis. system for allowing rapid, holistic, quantitative protein profiling. modifications. Optimizing sample preparation can nitrocellulose or PVDF membrane. The membrane My major area of interest and study has been to address the Some interesting technology is being developed (e.g., protein help minimize many of these obstacles. For example, is then incubated with an antibody specific for the inefficiencies of traditional two-gel separations for analyzing microarrays and ICAT), although each new approach comes with the extraction method and buffer can have a large protein of interest. An indirect method of detection, membrane proteins and other hydrophobic proteins. My research its own set of drawbacks. Despite its own limitations, the 2-D effect on the yield and quality of the sample and such as a secondary antibody-enzyme conjugate, is has shown that by applying prefractionation and improved gel approach coupled with advances in image analysis is still the components of interest. Nonprotein contaminants used to produce a recordable signal at the site of solubilization methods, proteins previously considered intractable most reliable for day-to-day use. that are extracted along with the proteins may be antibody binding. to 2-D electrophoresis are indeed amenable to the process. removed by size exclusion chromatography. Complex Specific antibodies have other uses, for example During my postdoctoral studies I shifted direction by Selected Publications protein mixtures can often be fractionated in cell localization studies and in inhibition of investigating techniques for profiling phosphorylation states Molloy MP et al., Overcoming technical variation and biological variation in effectively using ion exchange resins that separate activity or function studies, which can help in of proteins using mass spectrometry and chemical modification , Proteomics 3, 1912–1919. (2003) proteins based on their charge. Samples that contain assessing the role of the recognized protein. approaches. Nowadays in the pharmaceutical setting, my focus Molloy MP, The challenge of industrializing proteomics, Nat Biotechnol 21, 597 (2003) a large amount of a contaminating protein, such as Separation science is also important for obtaining is centered on turning the "data" collected using proteomic VanBogelen RA and Molloy MP, Exploring the proteome: reviving emphasis albumin in serum samples, can be purified by affinity antibodies. Antibodies are often generated to a approaches into "information" to understand the molecular on quantitative protein profiling, Proteomics 3, 1833–1834 (2003) chromatography to remove the contaminating protein of interest, to one related to it, or to a physiology of our preclinical models and enhance the discovery Molloy MP and Witzmann FA, Proteomics: Technologies and applications, protein and to enrich less-abundant proteins. synthetic peptide based on sequence information. of new drugs. Brief Funct Genomics Proteomics 1, 23–39 (2002) Preparative electrophoresis is another approach A partially purified target protein is a requirement There are major differences in operating a proteomics lab Benndorf R et al., HSP22, a new member of the small heat shock protein in an academic as opposed to a pharmaceutical setting. The superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27), to sample preparation. The most powerful for each of these approaches. Obtaining specific J Biol Chem 276, 26753–26761 (2001) most striking difference is the requirement to operate as part of preparative-scale technologies are continuous- antibodies also typically requires separation of these Nouwens AS et al., Complementing genomics with proteomics: the membrane a larger team in an industrial setting. The studies are too complex elution electrophoresis and liquid-phase isoelectric antibodies from contaminating proteins. subproteome of Pseudomonas aeruginosa PAO1, Electrophoresis 21, focusing. Liquid-phase isoelectric focusing has the A partially purified protein can often be identified and labor-intensive for any one person to conduct efficiently. 3797–3809 (2000) additional advantage of maintaining the biological by sequencing, either by Edman degradation using Consequently, the process is broken down into functional Santoni V et al., Membrane proteins and proteomics: un amour impossible? activity of proteins. automated sequencing systems, or by mass groups, with total reliance on colleagues to contribute. One Electrophoresis 21, 1054–1070 (2000) of the most rewarding aspects is being involved in tying these Once samples have been fractionated and partially spectrometry of peptide digests. Although significant Wilkins MR et al., High-throughput mass spectrometric discovery of protein pieces together and contributing a sizable volume of novel post-translational modifications, J Mol Biol 289, 645–657 (1999) purified, the next step in the process of both proteomic investment in capital equipment and user training is information to the therapeutic area groups that allow them to Molloy MP et al., Extraction of membrane proteins by differential solubilization and drug discovery studies is typically identification required, even a partial sequence, combined with make new decisions. for separation using two-dimensional gel electrophoresis, Electrophoresis 19, and characterization of proteins of interest. isoelectric point and molecular weight, can A second major difference is the acceptance of proteomics as 837–844 (1998) positively identify a protein based on comparison to a powerful discovery-based discipline. We find that there is great Walsh BJ et al., The Australian Proteome Analysis Facility (APAF): assembling large scale proteomics through integration and automation, Electrophoresis Protein Identification and Characterization information in protein and nucleic acid databases. value in building protein response databases in a discovery- 19, 1883–1890 (1998) Rapid, accurate, and reliable methods of identifying This approach is often combined with 2-D analysis oriented mode, that then allow hypothesis-driven questions to a protein of interest are necessary in order to to provide positive identification of protein spots Molloy MP et al., Identification of wallaby milk whey proteins separated by be addressed. In academia it is more difficult to find support for two-dimensional electrophoresis, using amino acid analysis and sequence efficiently carry out further studies. Preliminary excised from a gel. These methods are extremely discovery-driven investments. tagging, Electrophoresis 18, 1073–1078 (1997) characterization includes molecular weight and accurate and can detect low levels of contamination isoelectric point estimation by 2-D gel electrophoresis, by other proteins. If the protein is not an exact

14 BioRadiations 112 BioRadiations 112 15 cover story cover story

and costs of the chromatography systems are the basic objectives of protein purification are the entirely different: At laboratory scale, a $30,000 same for both laboratory and process scales, the system occupying a space of 40 cm x 40 cm x 1 m considerations are very different. will be adequate for performing multistep purifications, whereas at the process scale, a system The Future of Protein Separation and column priced at $250,000 and occupying At any stage of studying proteins — from initial 1.5 m x 1.5 m x 2 m are sufficient for only one step sample preparation through characterization and of a purification. Process-scale purification also eventual purification — chromatography and requires validation to ensure a reproducible product, electrophoresis are complementary, reliable with the added burdens of compliance with techniques. Both can be used to fractionate regulatory agencies, compatibility with materials of complex samples for further analysis, facilitating Fig. 3. Relative scale of laboratory construction, and cost control. During scale-up, the identification and characterization of proteins of (left) and process (right) number of buffers needed, the buffer constituents, interest. Individual proteins can be studied to chromatography systems. and their impact on health and safety all become address questions surrounding their activities, to significant considerations. Imagine a scale-up of a profile diseases, or to assess their usefulness as drugs reverse-phase chromatography method using the or drug targets; these applications and others have organic modifier TFA. At the laboratory scale, contributed to the central role of these powerful 1% TFA is only 1 ml in 100 ml of buffer, but at the protein separation techniques in the fields of process scale it is 20 L in 2,000 L. Here, how to proteomics and drug discovery. As these fields handle 20 L of TFA, and the costs of disposing of expand, the application of protein separation 2,000 L of buffer containing 1% TFA, become techniques will lead to new areas of investigation, Protein identification methods have other uses Several strategies are available for purification. critical concerns. Special precautions must be improve diagnosis and treatment of disease, and besides those in proteomics and drug discovery. Chromatography is currently the predominant established for operators, and disposal is regulated. drive growth in the basic research, biotechnology, These methods can often be used as diagnostic tools technique for preparing sufficient amounts of a Now a little TFA becomes a big problem. Although and pharmaceutical industries. for early detection of disease. For example, elevated purified target protein. It also exploits more cardiac enzymes, detected and quantitated by physicochemical properties to resolve target electrophoresis, are indicative of heart damage. molecules and contaminants, including Protein Separation: A Career Worth Pursuing Levels of two proteins found in a recent study may hydrophobicity and specific affinity (see Table). A one day serve as markers of Alzheimer disease and purification scheme generally combines two or more Thoughts and Career Advice help doctors diagnose and treat the disease earlier techniques to yield effective separation. At each Duncan Low, Senior Research Fellow, Amgen than with currently available methods (Sunderland step, suitable yield and purity must be obtained. Who ever thought a great career was easy? It his or her entire career. Imagine the challenge of engineering proteins T et al., Decreased beta-amyloid1-42 and increased Initially, experimentation may be required to continues to challenge me even after 25 years in that are ultimately used as vaccines in rural Africa, or developing tau levels in cerebrospinal fluid of patients with evaluate the effectiveness of different approaches. In the business. I first joined the industry in Europe manufacturing processes for a drug to treat arthritis. Alzheimer disease, JAMA 289, 2094–2103, 2003). addition to a wide range of standard media to select in the late 1970s as a product manager for ion Your Market Value from, the buffer composition can be modified for exchange chromatography, after finishing my If you are at the beginning of your studies, opt for rigor. Early Protein Purification better separation of the protein and contaminants. PhD in Microbiology from the University of training in mathematics and basic sciences will provide many future Obtaining a protein free of contaminants that Examples include addition of different buffering Glasgow. Later, I managed subsidiary offices options. Summer internships at biotechnology companies will in Australia, the UK, and the USA, each position deepening my interfere with its intended use is the objective of both agents, salts, detergents, zwitterions, and so on. expose you to the trials of real-life science and make you more understanding of the needs of the biotech industry. I recently laboratory- and process-scale purification strategies. Further tailoring of the purification strategy may valuable to future employers. Learn computer modeling, which will joined Amgen, one of the largest biotech companies in the world. In the laboratory, protein purification is a means of also be possible based on known characteristics of become a required job skill much as word processing and My ambition now is to to continually improve the level of science producing a suitable sample for both antibody the protein. Size and charge are obvious examples, spreadsheet applications have. generation and X-ray crystallography. Purification is but sequence information can also be exploited for at Amgen and to attract investors and “the best and the also useful for evaluation of protein function. At the both purification and further characterization. brightest” people to continue our successful track record in Nourish Your Passion process scale, the main objective is to produce large Overproduction of a cloned protein with a suitable biopharmaceuticals. Last but not least, continue to learn new information and skills. amounts of purified protein that meet rigorous quality expression system allows its enrichment and Jobs and Money Constant learning and exposure to new ideas is the difference between an excellent and a mediocre career. standards, generally to meet known market needs, potentially higher yield and purity. Cloning also If your ambition is to obtain a challenging, well-paid job such as production of a pharmaceutical. allows the inclusion of sequence modifications or with opportunity for travel and learning over an entire career, consider biopurification. There are many positions at top-level 9,000 250 additions that facilitate either purification (such as Companies a His-tag; see article on page 6) or evaluation of biotechnology companies worldwide that offer laboratories with 8,000 Patents Table. Examples of target proteins and the properties useful in their separation. Jobs (thousands) function. Sequence information may suggest ligands the latest tools for R&D and process development. According to 7,000 Biotech approvals 200 Target and Contaminants Molecular Characteristic Method and the Biotechnology Industrial Organization (www.bio.org) and to Exploit Chromatographic Support that can be exploited in purification. Antibodies are 6,000 other sources, key indicators such as the numbers of jobs and 150 Monoclonal antibody (Mab) from Ca2+ coordination Coordination chemistry, occasionally useful ligands, although their cost is 5,000 Mab and protein A aggregate ceramic hydroxyapatite drug approvals in the U.S. have continued to rise throughout usually prohibitive for large-scale purification. 4,000 the recent market downtrend (see figure). 100 Target from trace Hydrophobicity Hydrophobic interaction, At laboratory scale, several techniques can be 3,000 E. coli proteins Macro-Prep t-butyl evaluated for their effectiveness, then scaled up and Challenges and Choice Jobs & Approvals Patents & Companies 2,000 50 modified as appropriate for a larger-scale Commercialization of bioengineered products is a very complex route His-tagged protein capture from Affinity for metal ions Affinity, UNOsphere IMAC 1,000 other cellular proteins purification. Modification is often necessary because from discovery to large-scale manufacturing that offers an abundance 0 0 Mab capture from high volumes Charge Ion exchange, UNOsphere S laboratory-scale chromatography can differ radically of career positions, all benefiting from comprehensive training in the 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 of cell culture fluid principles of biopurification. The average scientist entering Approval data courtesy of Biotechnology Industry Organization. Job and company from process chromatography. At laboratory scale, data courtesy of Ernst & Young’s annual biotechnology report. Patent data source, Albumin monomer from Size Size exclusion, Bio-Gel P-100 packing a column is trivial, while at a large scale it biotechnology today will hold more than 15 different positions over US Patent and Trademark Office. albumin aggregates may require an overhead hoist. Space requirements

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exposure of personnel to radiation, high disposal different colored fluorophores allows simultaneous costs, and environmental concerns. Lastly, detection of several target proteins on the same blot. chemiluminescent methods allow stripping and reprobing of blots multiple times, which is not Autoradiography possible with colorimetric detection techniques. Radioisotopes can be used to label probes (in the Western Blot Detection Methods case of western blots, the secondary antibodies) for Bioluminescent Detection most blotting applications. The radioisotopes Introduction phosphor imagers. CCD systems also offer Bioluminescence is the phenomenon of light commonly used in biological sciences include 35S, Western blotting is a routine yet powerful tool for convenience and efficiency by providing a digital emission by many organisms. Bioluminescent 32P, 33P, 14C, and 125I. To detect the radioactivity, A. Chemiluminescence detection and characterization of proteins. The record of experiments for sharing and data analysis systems differ in the structure and function of the the most widely used method is autoradiography technology takes advantage of the specificity of and by eliminating the need to continually enzymes and cofactors involved in the process as on X-ray film. Autoradiography provides a good antibodies to provide definitive identification of purchase consumables for film development. well as the mechanism of the light-generating combination of sensitivity and resolution without proteins that have been separated by polyacrylamide Chemiluminescent technology is easily adapted reactions. a large investment. For direct autoradiography gel electrophoresis and transferred to a membrane. to existing western blotting procedures because it Bioluminescence can be exploited as a detection without intensifying screens or scintillators, the Western blotting involves sequential recognition uses antibody-conjugated enzymes to generate the method for western blots. Bioluminescent detection response of the film is linear only within a range of between three components: the target protein, a light signal. The blocking and wash procedures involves incubation of the membrane with the 1–2 orders of magnitude. When intensifying screens primary antibody that recognizes the target protein, are familiar western blotting steps. Most chemi- bound antigen/antibody-enzyme complex in a or fluorographic scintillators are used to increase and a secondary antibody that both recognizes the luminescent methods require only a few chemical bioluminogenic substrate and the simultaneous sensitivity, the response of the film is nonlinear, but primary antibody and generates a detectable signal. components to generate light. For luminol chemi- measurement of emitted light. The substrate in it can easily be made linear by preexposing the film B. Bioluminescence After proteins have been bound to a membrane, luminescence, a substrate is chemically oxidized by this detection system is a luciferin-based derivative. to a flash of light. the membrane is blocked, incubated with primary HRP to produce an excited-state product. In most Light detection is performed using a photon- Phosphor imagers such as Bio-Rad’s Molecular antibody, then secondary antibody, and washed. The luminol-based methods, including the Immun-Star™ counting camera, and the blotted proteins are Imager FX™ family of imagers offer an alternative primary antibody binds to the protein of interest, HRP kit, light is emitted when the excited product visualized as bright spots. This technique is very to film detection methods. The initial investment and the secondary antibody enables its detection. returns to the ground state. The signal of the blot is similar to chemiluminescence in sensitivity and in instrumentation offers increased sensitivity The secondary antibody can be labeled with a visualized by exposing the blot to film or using speed of detection but is not as widely used, and and dynamic range compared to X-ray film, and radioisotope, a fluorophore, or an enzyme, typically imaging systems as described above. Besides luminol, few bioluminogenic substrates are commercially exposure times are 10 to 20 times shorter than those horseradish peroxidase (HRP) or alkaline some other commercially available chemiluminescent available. PVDF is the preferred membrane for for film. The ability to accurately quantitate data is phosphatase (AP). substrates include acridan-based substrates like bioluminescent detection because nitrocellulose also much greater with storage phosphor screens For many years, radioisotopes were the method Lumigen PS-3 (which use AP or HRP as a reporter membranes may contain substances that inhibit because the linear dynamic range of phosphor C. Chemifluorescence of choice to label secondary antibody probes for enzyme) and 1-2-dioxetane-based substrates (which luciferase activity and thus interfere with the assay. imagers is significantly greater, 4.8 orders of blotting applications. Newer methods are less use AP as a reporter enzyme). The emission of light magnitude. The advantage of a linear dynamic hazardous and easier to use than radioactivity, can be enhanced up to 1,000-fold by the addition Chemifluorescent Detection range is that there is a direct relationship between but give comparable sensitivity. Current western of enhancers such as phenols. The enhancer used Chemifluorescence is the enzymatic conversion signal intensity and actual quantity. This enables blotting methods include light-based (chemi- differentiates one supplier’s kit from another. of a substrate to a fluorescent product. Fluorogenic accurate quantitation and the elimination of luminescent, bioluminescent, chemifluorescent Chemiluminescent western blot detection has compounds (nonfluorescent or weakly fluorescent overexposure and saturated signals. and fluorescent) detection, autoradiography, and several advantages over other methods, including substances that can be converted to fluorescent colorimetric detection (Table 1 and Figure 1). speed and sensitivity. Average exposure times are 30 products) are available to use with a wide variety Colorimetric Detection seconds to 15 minutes. This is a large improvement of enzymes, including AP and HRP. The enzyme Several substrates are converted to a colored D. Fluorescence/autoradiography Chemiluminescent Detection over 125I detection, which can require up to 48 hours cleaves a phosphate group from a fluorogenic precipitate by enzymes such as HRP or AP, which are Chemiluminescence is the production of detectable of film exposure. Detection of low picogram amounts substrate to yield a highly fluorescent product. The usually conjugated to the secondary antibody. The light from a chemical reaction. For western blot of protein is typical of chemiluminescent systems, fluorescence can be detected using a fluorescence most commonly used substrates include 5-bromo-4- detection, such reactions can be catalyzed by an which are more sensitive than most colorimetric imager such as the Molecular Imager FX™ Pro Plus chloro-3-indolyl phosphate/Nitroblue Tetrazolium enzyme such as AP or HRP conjugated to an systems, and approximately equal to that of system or VersaDoc system and quantitated using (BCIP/NBT) for AP and diaminobenzidine (DAB) antibody. The light signal can be captured on X-ray radioisotopic detection. It is important to note Quantity One® software. Chemifluorescence can or 4-chloro-1-naphthol (4CN) for HRP. As the film or on CCD instruments such as the Bio-Rad that the detection sensitivity is somewhat dependent provide a stable fluorescent reaction product, so precipitate accumulates, a visible colored signal VersaDoc™ and ChemiDoc™ XRS systems. on the affinity of the antigen and primary and that blots can be scanned at a convenient time. develops on the blot. The enzyme reaction can be The benefits of detecting chemiluminescent secondary antibodies and can vary considerably from The method is compatible with standard stripping monitored and stopped when the desired signal-to- E. Colorimetric detection signals with a CCD system are numerous. The one protein sample to another. and reprobing procedures. noise level is reached. The method is easier than any linear dynamic range of CCD systems is 2 to 4.8 Chemiluminescent detection does not have the orders of magnitude, comparable to that of disadvantages related to isotope detection, such as Fluorescent Detection Target protein In fluorescent detection, the secondary antibody Fig. 1. Mechanisms of detection method chemistries. Table 1. Comparison of western blot detection methods. is labeled with a fluorophore such as fluorescein In each method of western blot detection, a detectable signal Primary antibody is generated following binding of an antibody specific for the Luminescent (FITC), Texas Red, rhodamine (TRITC), or (Chemi- and Bio-) Chemifluorescent Fluorescent Radioisotopic Colorimetric protein of interest. Chemiluminescent, bioluminescent, and Secondary antibody R-phycoerythrin. The main advantage of fluorescent chemifluorescent detection all rely on generation of a light signal. Sensitivity Excellent Very good Very good Excellent Very good detection is that it can provide a 10-fold greater In chemiluminescent (A) and bioluminescent (B) detection, the Economy of antibody use Excellent Very good Excellent Excellent Good Enzyme conjugate linear dynamic range with only 2–4-fold reduced reaction itself emits light. Chemiluminescent and bioluminescent Speed of detection Excellent Excellent Excellent Poor Good detection are distinguished by the source of the substrate. In Substrate Ease of reprobing Very good Fair Fair Fair Poor sensitivity over chemiluminescent detection. chemifluorescent detection (C), the product is fluorescent. Both Ease of quantitation Very good Fair Excellent Excellent Fair Fluorescent western blot detection can therefore fluorescent detection and autoradiography (D) record the signal Product generated by a labeled secondary antibody. In fluorescent Durability of results Excellent Fair Excellent Excellent Good provide better linearity and better quantitation detection, the antibody is labeled with a fluorophore, while in Label (radiolabel or fluorophore) Suitable detection system ChemiDoc XRS Molecular Imager Molecular Imager Molecular Imager GS-800 within the detection limits. Fluorescent detection autoradiography, it is labeled with a radioactive isotope. In Emitted light or radiation VersaDoc 3000/4000 FX Pro Plus FX Pro Plus FX Pro Plus also allows multiplexing. Multiplexing with colorimetric detection (E), the signal is a colored precipitate.

18 BioRadiations 112 BioRadiations 112 19 dimensions tips and techniques

film-based detection method, which must be radioisotopic or chemiluminescent detection. developed by trial and error, and it doesn’t use costly However, Bio-Rad has colorimetric systems that materials such as X-ray film and darkroom chemicals. offer very high sensitivity matching that of Colorimetric samples can be easily recorded and chemiluminescence. These include the Immun-Blot® analyzed with a densitometer such as the GS-800™ amplified AP assay kit, which uses a biotinylated calibrated densitometer. The densitometer provides secondary antibody and a streptavidin-biotinylated Standard Curves as Troubleshooting Tools for a digital record of the blot, excellent resolution, AP complex to amplify the signal, and the HRP- reproducible results, and accurate quantitation. based amplified Opti-4CN™ kit. Real-Time RT-PCR Assays The GS-800 also uses red-, green-, and blue-color CCD technology to greatly improve the detection Bio-Rad offers many products for blotting and blot Real-time PCR is a powerful technique with a broad concentration of cDNA. Additionally, the efficiency of a wide range of colorimetric detection reagents. detection. For more information, request Bio-Rad’s range of applications. The most common application of the assay derived from the slope of the standard Colorimetric detection is typically considered western blotting products folder (bulletin 2033) or is evaluating mRNA levels by reverse transcription curve is 121% — 21% higher than the theoretical a medium-sensitivity method compared to visit us on the Web at discover.bio-rad.com PCR (RT-PCR). This is also one of the more limit. Both of these factors indicate a problem in the challenging applications, as it involves two distinct experimental system. Ordering Information enzymatic reactions — reverse transcription and PCR Excluding the problematic 100 ng standard from Catalog # Description Catalog # Description — and the use of internal controls that substitute for the data set allows the other four standards to fall on Immun-Star HRP Chemiluminescent Detection Kits Blotting Membranes (cont.) the more ideal normalization to cell number. a straight line, and the derived efficiency is now a 170-5040 Immun-Star HRP Substrate, 500 ml 162-0236 Sequi-Blot™ PVDF/Filter Paper Sandwiches, 170-5041 Immun-Star HRP Substrate, 100 ml 8.5 x 13.5 cm, 20 pack This article focuses on standard curves generated more acceptable 101% (Figure 1C). 170-5042 Goat Anti-Rabbit-HRP Detection Reagents 162-0237 Sequi-Blot PVDF/Filter Paper Sandwiches, from different templates available to the researcher. Results of this kind, in which one or more 170-5043 Goat Anti-Mouse-HRP Detection Reagents 8.5 x 13.5 cm, 50 pack Using different templates to evaluate real-time PCR standards do not fall on a straight line, are not 170-5044 Goat Anti-Mouse-HRP Detection Kit 162-0212 0.2 µm Nitrocellulose/Filter Paper Sandwiches, assays can provide valuable information, even if your uncommon and are usually the result of some kind of 170-5045 Goat Anti-Rabbit-HRP Detection Kit 7 x 8.5 cm, 20 pack Immun-Star AP Chemiluminescent Detection Kits 162-0213 0.2 µm Nitrocellulose/Filter Paper Sandwiches, experimental design does not require that standard inhibitor present in the cDNA sample. The inhibitor 170-5018 Immun-Star AP Substrate 7 x 8.5 cm, 50 pack curves be run. is diluted out at the lower concentrations, so it does 170-5010 Goat Anti-Mouse-AP Detection Kit 162-0214 0.45 µm Nitrocellulose/Filter Paper Sandwiches, Standard curves derived from serial dilutions not affect the kinetics of the experiment at these 170-5011 Goat Anti-Rabbit-AP Detection Kit 7 x 8.5 cm, 20 pack of samples provide a useful tool to evaluate the concentrations. The fact that this results in >100% 170-5012 AP Substrate Pack 162-0215 0.45 µm Nitrocellulose/Filter Paper Sandwiches, consistency of these enzymatic reactions. These efficiency may be counterintuitive, but remember 170-5013 Goat Anti-Mouse-AP Intro Kit 7 x 8.5 cm, 50 pack 170-5014 Goat Anti-Rabbit-AP Intro Kit 162-0218 Immun-Blot PVDF/Filter Paper Sandwiches, experiments test the response of your reagent that the efficiency value is derived from the slope of Fig. 1. Evaluation of 170-5015 Blotting Reagents Pack 7 x 8.5 cm, 20 pack system to different starting quantities. Similar to the standard curve. The inhibitor is causing the high- assay results based Immun-Blot AP and HRP Colorimetric Assay Kits 162-0219 Immun-Blot PVDF/Filter Paper Sandwiches, a mathematical formula, the assay should return concentration standard to have a higher C value on standard curves. T A, serial dilutions of 170-6460 Goat Anti-Rabbit IgG (H + L)-AP Assay Kit 7 x 8.5 cm, 50 pack predictable and consistent results based on the than it should. This makes the slope shallow, thus 170-6461 Goat Anti-Mouse IgG (H + L)-AP Assay Kit 162-0216 Sequi-Blot PVDF/Filter Paper Sandwiches, plasmid template from 108 to 102 copies. B, 170-6462 Goat Anti-Human IgG (H + L)-AP Assay Kit 7 x 8.5 cm, 20 pack inputs. The equation for the standard curve is in resulting in a derived efficiency over 100%. It is often serial dilutions of cDNA 170-6412 Immun-Blot Amplified AP Assay Kit 162-0217 Sequi-Blot PVDF/Filter Paper Sandwiches, fact a mathematical function describing the assay necessary to examine the traces of the real-time representing 100 ng to 170-6463 Goat Anti-Rabbit IgG (H + L)-HRP Assay Kit 7 x 8.5 cm, 50 pack in question. amplifications (not shown) and assess the slopes to 10 pg of input RNA. C, 170-6464 Goat Anti-Mouse IgG (H + L)-HRP Assay Kit 162-0177 Immun-Blot PVDF Membrane, 26 cm x 3.3 m Figure 1A shows an example of a standard curve determine if the individual efficiencies are serial dilutions of cDNA 170-6465 Goat Anti-Human IgG (H + L)-HRP Assay Kit 162-0184 Sequi-Blot PVDF Membrane, 24 cm x 3.3 m run with a 100-fold dilution series, from 108 to 102 approximately equivalent. All traces should be described in B with 170-8238 Amplified Opti-4CN Substrate Kit 162-0115 Nitrocellulose Membrane, 0.45 µm, 30 cm x 3.5 m copies, of plasmid DNA. The fact that the curve is 100 ng standard 170-8235 Opti-4CN Substrate Kit 162-0112 Nitrocellulose Membrane, 0.2 µm, 30 cm x 3.5 m excluded. Premixed Substrate Reagents 162-0094 Supported Nitrocellulose Membrane, 0.45 µm, linear demonstrates that the assay has responded 2 170-6431 HRP Conjugate Substrate Kit 30 cm x 3 m consistently to all concentrations of template tested. A r : 0.998 Slope: –3.395 Intercept: 37.543 Y = –3.395X + 37.543 170-6432 AP Conjugate Substrate Kit 162-0095 Supported Nitrocellulose Membrane, 0.2 µm, PCR efficiency: 97.0% The equation for the curve is the mathematical 34 Blot Transfer and Processing Buffers 30 cm x 3 m Unknown 30 161-0778 10x Tris/CAPS, 1 L Blotting Apparatus function, and the red line is a graphical 26 Standards ® representation of that function. The fact that all of 161-0774 20x SSC, 1 L 170-3930 Mini Trans-Blot Electrophoretic Transfer Cell 22 161-0780 10x PBS, 1 L 170-4070 Criterion™ Blotter With Plate Electrodes the standards fall on the curve indicates that the 18 170-6435 10x TBS, 1 L 170-4071 Criterion Blotter With Wire Electrodes assay is returning the expected threshold cycle (C ) 14

® T cycle Threshold 10 161-0781 10% Tween 20, 1 L 170-3939 Trans-Blot Cell With Plate Electrodes values for the tested input quantities. r2 is the 12345678 170-6531 Tween 20, EIA grade, 100 ml 170-3940 Trans-Blot SDSemi-Dry System log Starting quantity, plasmid copy number 161-0783 1x Phosphate Buffered Saline With 1% Casein, 1 L Power Supplies correlation coefficient squared and is a measure of 161-0782 1x Tris Buffered Saline With 1% Casein, 1 L 164-5052 PowerPac™ HC Power Supply how closely the calculated C values fit the expected T B r2: 0.980 Slope: –2.903 Intercept: 31.963 Y = –2.903X + 31.963 Blotting Membranes 164-5050 PowerPac™ Basic Power Supply 2 values. r is a positive number, and the closer to PCR efficiency: 121.1% 162-0232 0.2 µm Nitrocellulose/Filter Paper Sandwiches, Imaging Equipment 1.000, the better the fit. 8.5 x 13.5 cm, 20 pack 170-7850 Molecular Imager FX Pro Plus System, PC 162-0233 0.2 µm Nitrocellulose/Filter Paper Sandwiches, 170-7851 Molecular Imager FX Pro Plus System, Mac Even if you are using cDNA, this type of 8.5 x 13.5 cm, 50 pack 170-8030 VersaDoc Model 3000 Imaging System, PC experiment may be important to run because a 162-0234 0.45 µm Nitrocellulose/Filter Paper Sandwiches, 170-3031 VersaDoc Model 3000 Imaging System, Mac plasmid DNA template has none of the confounding 8.5 x 13.5 cm, 20 pack 170-8140 VersaDoc Model 4000 Imaging System, PC factors that might be present in samples isolated –2 –1 0 1 2 162-0235 0.45 µm Nitrocellulose/Filter Paper Sandwiches, 170-8141 VersaDoc Model 4000 Imaging System, Mac from an experimental system, reverse transcribed, log Starting quantity, ng input RNA 8.5 x 13.5 cm, 50 pack 170-7980 GS-800 Calibrated Densitometer, PC 162-0238 Immun-Blot PVDF/Filter Paper Sandwiches, 170-7981 GS-800 Calibrated Densitometer, Mac and stored over a period of time. 2 8.5 x 13.5 cm, 20 pack 170-8070 ChemiDoc XRS System, PC Running the experiment on a dilution series of C r : 0.996 Slope: –3.292 Intercept: 31.574 Y = –3.292X + 31.574 162-0239 Immun-Blot PVDF/Filter Paper Sandwiches, 170-8071 ChemiDoc XRS System, Mac cDNA provides you with additional information. PCR efficiency: 101.3% 8.5 x 13.5 cm, 50 pack Background Remover Figure 1B is an example of one such assay. This 10- ™ 170-5020 DeExpose Background Remover, 10x, 250 ml fold dilution series of cDNA represents 100 ng down to 10 pg of RNA. Note that the 100 ng standard does

not fall on the standard curve. The assay is not –2 –1 0 1 returning the expected CT value for that log Starting quantity, ng input RNA

20 BioRadiations 112 BioRadiations 112 21 tips and techniques tips and techniques

parallel when viewed with the y-axis on the log scale. or gel-isolated PCR product to evaluate the assay. Possible sources of inhibition are organic solvents Comparing the results of serial dilutions of clean, carried over from RNA isolation, inhibitory proteins homogeneous samples like purified plasmid to not removed during isolation, and ethanol left from representative cDNA samples can yield valuable the precipitation or wash steps. It is also important information about your assay. The plasmid experiment Prevention of Vertical Streaking to heat-inactivate the reverse transcriptase, as it too reflects the true kinetics of an assay without the will inhibit your PCR reaction. confounding factors that can be introduced with Tom Berkelman, Mary Grace Brubacher, and Haiyin Chang, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA cDNA. The serial dilutions of representative cDNA How Does This Apply to Your Research? demonstrate the performance of the assay in the An important and often overlooked consideration real-world experiment, namely samples from the In the last issue of BioRadiations (111), we discussed A B is evaluating the PCR reaction without the biological system being studied. Running only the the various causes of horizontal streaking on 2-D confounding factors presented by the biological cDNA experiment may lead you to redesign a gels, and its prevention. Horizontal streaking is the sample. In addition to being heterogeneous, cDNA perfectly acceptable assay when sample preparation result of problems that occur during the first- samples are obtained from sources that may contain is the only problem. Performing the plasmid dimension separation (generally performed in IPG inhibitors. The isolation process itself may also experiment tests the assay in ideal conditions. strips). This article focuses on vertical streaking, introduce inhibiting compounds. In some cases the There is one more serial dilution that should be which results from problems related to second- effect of these impurities is not as clear as in the performed to assess your experimental conditions. So dimension separation. Vertical streaking should not example given. Results may not be reproducible at far the discussion has focused on the PCR reaction’s be confused with vertical gaps or blank regions in any concentration. There may be no three response to different sample sources. The other the 2-D pattern (see final paragraph). Examples of Fig. 1. Examples of vertical streaking. consecutive standards that actually show a linear enzymatic reaction in RT-PCR-based expression vertical streaking are presented in Figure 1. A, approximately 300 µg of E. coli protein loaded onto a 17 cm pH 3–10 ReadyStrip™ relationship. When results like these are obtained, analysis is the reverse transcription (RT) reaction. IPG strip. B, a prefractionated sample of the next logical step is to use a high-purity plasmid This reaction can be conceptualized as a simple Poor Protein Solubility Pseudomonas putida loaded onto a 17 cm enzymatic reaction involving an enzyme, reverse Following the first-dimension separation, all of the pH 3–10 ReadyStrip IPG strip. Gel image was transcriptase, and mRNA as the substrate, though proteins have moved to their isoelectric points and annotated using The Discovery Series™ A Serially diluted RNA PDQuest™ 2-D analysis software. Fig. 2. Experimental it does not fit the classical definition of a substrate. carry no net charge. The ionic strength of the medium evaluation of reverse 1 µg The cDNA is clearly the product of interest. This is also very low, because small ions have all migrated transcription (RT) reactions. 100 ng enzymatic reaction can be saturated and does have out of the first-dimension strip. Proteins generally have Ineffective Equilibration A, serial dilutions of RNA were prepared and reverse 10 ng a limit of detection. Therefore, you should test these minimal solubility under these conditions and are Equilibration should be carried out in a manner transcribed, followed by Real-time parameters of the RT reagent system with varying often precipitated within the gel matrix despite the that ensures good penetration of SDS and therefore quantitation of the resulting 1 ng RT PCR concentrations of RNA to verify that the system is presence of detergents, chaotropic agents, and other protein solubilization. The equilibration tray should cDNA by real-time PCR. B, 100 pg reverse transcribing RNA to cDNA reproducibly solubilizing additives. The major purpose of the be shaken or rocked to ensure continual movement after reverse transcription of 1 µg RNA, the resulting cDNA 10 pg and predictably. A dilution series of RNA is equilibration step following the first dimension is to of solution. Equilibration may be prolonged to as was serially diluted and prepared, and then each of these dilutions is reverse coat the proteins with SDS, giving them a negative long as 45 minutes if necessary. This may result in quantitated by real-time PCR. 1 pg transcribed (Figure 2A). The proportion of cDNA charge and making them soluble again so that they the loss of some small polypeptides (<15 kD), but Ghosted values indicate transcript produced from each RNA dilution should may enter the second-dimension gel. loss of larger proteins is generally insignificant theoretically equivalent B Serially diluted cDNA amounts of starting RNA. C, be the same. even with prolonged equilibration. SDS should be the resulting standard curves 1 µg RT 1 µg The resulting cDNA samples are then analyzed High Protein Load present at a concentration of at least 2% (w/v), were essentially the same. 100 ng using a real-time PCR assay. The resulting standard When a large quantity of protein is loaded, or when particularly if high (>2%) concentrations of 10 ng curve should be linear and have the same slope the sample contains proteins of particularly high detergent were used in the first dimension. Other (efficiency) as a curve produced by a dilution series abundance, resolubilization during equilibration may components of the equilibration solution are 1 ng Real-time PCR of cDNA (Figure 2B). Figure 2C demonstrates the be incomplete, resulting in vertical streaking and important as well. The solution should be buffered, 100 pg results of such an experiment performed to test tailing of the most intense protein spots. This can be particularly if an alkylation step is employed, ™ 10 pg Bio-Rad’s iScript cDNA synthesis kit. prevented by limiting the amount of protein loaded because iodoacetamide treatment generates acid. Whether you are evaluating the PCR reaction onto the first-dimension strip. It is often possible to The optimal pH for equilibration is 8.8. Glycerol, 1 pg or the RT reaction, the information obtained from compensate for a lower protein load by using a more which should be present at a concentration of at standard curves can be used to make decisions about sensitive staining technique (for example, silver least 20% (v/v), is a particularly important C 40 experimental design. Only concentrations that stain, catalog #161-0449, or SYPRO Ruby protein component of the equilibration solution, and its ■ cDNA ▲ Total RNA produce linear standard curves should be used, and gel stain, catalog #170-3125, instead of Coomassie omission will result in vertical streaking. 35 aberrant slopes or efficiencies should be investigated. Blue stain). In some cases, vertical streaking of ■ ▲ The RT reaction in particular is susceptible to abundant proteins can be prevented by prolonging Protein Oxidation 30 ■ ▲ saturation and sensitivity issues. The PCR reaction, the equilibration time (see “Ineffective Oxidative crosslinking or protein refolding should be ■ 25 ▲ though usually not susceptible to saturation, may Equilibration” below). prevented during all steps of the 2-D process; protein ■ ▲ be inhibited by contaminants. Generating standard oxidation during the second dimension separation 20 ■ ▲ curves allows you to evaluate whether these factors Overfocusing can result in vertical streaking. Treatment with the ™ Threshold cycle Threshold 15 cDNA Total RNA ■▲ are likely to become problematic, and at what Isoelectric precipitation increases with focusing time, ReadyPrep reduction-alkylation kit (catalog #163- ■ r2 0.998 0.998 ▲ concentration. You can then proceed with an so vertical streaking may be minimized by ensuring 2090) prior to first-dimension focusing can block 10 Slope –3.394 –3.324 appropriate strategy with the experimental samples that first-dimension IEF is not conducted for any cysteine sulfhydryl groups and prevent their Intercept 38.91 38.09 of your system of interest. longer than necessary. reoxidation. If the sample is not alkylated, alkylation 5 123456789 should be performed in a second equilibration step log Starting quantity, fg input RNA with 2.5% iodoacetamide. If alkylation is not

22 BioRadiations 112 BioRadiations 112 23 tips and techniques

desired, a sufficient quantity of dithiothreitol (DTT), is pressed firmly against one of the second-dimension at least 1%, should be present in the equilibration gel plates. An agarose overlay should be used to solution. The DTT should be added to the prevent the IPG strip from coming loose or moving. equilibration solution immediately before use, as it Bubbles in the agarose overlay should be minimized. may oxidize if stored. DTT is negatively charged under second-dimension conditions and will migrate Uneven Pore Size in the IPG Strip ahead of proteins in the gel, ensuring a reducing Following IEF environment during the separation. After the first-dimension separation, there are often variations in the thickness of the IPG strip. This Poor Reagent Quality or Improperly swelling or thinning of some sections is caused by Prepared Solutions water movement in the gel (Rabilloud 2000). In Vertical streaking and other second-dimension thinner sections of the IPG strip, the gel pore size problems often result from poor reagent quality or may have been reduced to the point that protein from mistakes in solution preparation. Care should migration out of the IPG strips will be slowed or be taken to use the highest-quality reagents, and the prevented. This can result in vertical streaks or, in pH of all buffers should be verified. Precast second- severe cases, complete protein loss from trapping dimension gels should be used before their within the IPG strip. Water movement during first- expiration date. Poor-quality acrylamide or old dimension separation can be minimized by ensuring acrylamide stock solutions can cause vertical that the sample is well desalted and that focusing is streaking due to an incompletely polymerized not carried out any longer than necessary. second-dimension gel. Reusing electrophoresis running buffer can result in poor separation and Vertical Gaps or Vertical Blank Stripes vertical streaking due to the depletion of ions and Vertical gaps or blank stripes, for the purposes of this SDS in the running buffer. This practice should be discussion, are different from vertical streaking. avoided if vertical streaking is a persistent problem. These problems can be due to trapped air bubbles in the agarose overlay on the IPG strip, excessive DTT Poor Placement of the IPG Strip on the (>50 mM) in the IPG sample buffer, or a compromised Second-Dimension Gel IPG strip (either insufficiently rehydrated or torn Vertical streaking can also be the result of gaps during handling). Blank stripes near the electrode, between the IPG strip and the second-dimension gel especially the cathode, can be caused by a buildup or damage to the IPG strip during application. The of salt. For further information on troubleshooting second-dimension gel should have a straight, level vertical gaps and other 2-D related problems, refer to top edge, and care should be taken that the IPG bulletin 2651 or contact us at consult.bio-rad.com strip is in direct contact with the second-dimension gel along its entire length. The IPG strip should not Reference be twisted during placement. This can be prevented Rabilloud T (ed), Proteome Research: Two-Dimensional Gel by ensuring that the plastic backing of the IPG strip Electrophoresis and Identification Methods, Springer-Verlag, p. 104 (2000) Timeless Beauty. With up to a 12-month shelf life, new Criterion™ XT gels new literature give you beautiful results any time. • Protein separations are exquisitely sharp General • Bio-Rad 2004/05 life science research products catalog • Gene Delivery to Mammalian Cells, edited by William C Heiser, is • The convenience of room temperature storage • 2004 Biotechnology Explorer™ educational products catalog a two-volume collection of step-by-step protocols for gene transfer that is part of the Methods in Molecular Biology series • A large sample capacity, combined with fast runs, Amplification published by Humana Press. Topics in volume 1 (ISBN 1-58829- enables impressive output 086-7) include ultrasound, peptides, DNA clamps, liposomes, • iTaq™ SYBR® Green supermix with ROX flier (bulletin 3065) microinjection, electroporation, particle bombardment, ™ • iScript one-step RT-PCR kit flier (bulletin 3066) dendrimers, and hydrodynamics. Robert S King contributed the • Preservation of protein stability during the run chapter on microinjection. Volume 2 (ISBN 1-58829-095-6) details Gene Transfer virus-mediated gene transfer, including in vitro and in vivo • Criterion XT gels join a collection of coordinated Two new books by Bio-Rad employees have been published. applications and lentiviral vectors. These books are available for products including a blotter, high-throughput cell, • Micromanipulation in Assisted Conception: A User’s Manual and purchase from Humana Press (www.humanapress.com). Troubleshooting Guide, by Steven D Fleming and Robert S King, and ready-to-use XT buffers — a complete system (ISBN 0521648475) is published by Cambridge University Press. Electrophoresis and Blotting that lets you be as productive as you are creative It includes detailed descriptions of all common micromanipulation • Western blotting detection reagent brochure (bulletin 2032) systems used by in vitro fertilization laboratories. While aimed • Protein standards user guide (bulletin 2998) Visit us now at criterion.bio-rad.com to learn more. primarily at clinical embryologists, the book contains chapters on ™ equipment optimization and micropipet fabrication as well as • De-Expose background remover flier (bulletin 3027) transgenic and gene knockout mouse production. It will be of • De-Expose background remover promotional card (bulletin 3028) value to any laboratory performing gamete and embryo • Precision Plus Protein™ standard plugs promotional card micromanipulation. The book covers state-of-the-art techniques (bulletin 3036) and their optimization, including ICSI, and procedures such as assisted hatching and blastomere biopsy.

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