Mini-PROTEAN Precast Gels

Mini-PROTEAN Precast Gels Instruction Manual and Application Guide For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-4BIORAD (1-800-424-6723)

Table of Contents Section 1 General Information...1 1.1 Introduction...1 1.2 Mini-PROTEAN Precast Gel Specifications...2 1.3 Important Notes...3 1.4 Mini-PROTEAN Comb Configurations...3 Section 2 Setup and Basic Operation...3 2.1 Required Materials...3 2.2 Mini-PROTEAN Precast Gel Set-Up Overview...4 2.3 Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module...5 Section 3 SDS-PAGE...6 3.1 Introduction...6 3.2 Mini-PROTEAN TGX Gel Composition...7 3.3 Mini-PROTEAN TGX Gel Selection Guide...7 3.4 SDS-PAGE Buffers...8 3.5 Sample Preparation...8 3.6 Running Conditions...8 Section 4 Native PAGE...8 4.1 Introduction...8 4.2 Native PAGE Buffers...8 4.3 Sample Preparation...8 4.4 Running Conditions...8 Section 5 Buffers...9 Section 6 Total Protein Gel Stains for SDS-PAGE and Native PAGE Detection...10 Section 7 Troubleshooting...11 Appendix A Stock Solutions...13 Appendix B Total Protein Blot Stains...14 Appendix C Related Literature...14 Appendix D Ordering Information...15 D.1 Mini-PROTEAN TGX Precast Gels...15 D.2 Premixed Running and Sample Buffers...15 D.3 Individual Reagents...15 D.4 Total Protein Gel and Blot Stains...16 D.5 Immunoblot Detection...17 D.6 Immunoblot Detection Reagents...17 D.7 Blotting Membranes...18 D.8 Protein Standards...18 D.9 Equipment...18

Section 1 General Information 1.1 Introduction Mini-PROTEAN precast gels greatly simplify polyacrylamide gel electrophoresis. They are specifically designed for use with the Mini-PROTEAN Systems (Mini-PROTEAN Tetra, Mini-PROTEAN 3, and Mini-PROTEAN Dodeca Cells). Mini-PROTEAN precast gels come ready to use with pre-formed sample wells and a stacking layer. Each Mini-PROTEAN cassette is 8.5 cm x 10 cm (H x W) and 4.0 mm thick. Gel dimension is 7.2 cm x 8.6 cm (H x W) and 1.0 mm thick. Each gel is individually packaged in a leak proof storage pouch with gel buffer containing 0.02% sodium azide. The migration pattern of proteins on Mini-PROTEAN TGX precast gels is similar to that observed with standard Laemmli Tris-HCl gels. Mini-PROTEAN TGX precast gels are run using standard Laemmli sample buffer and Tris-Glycine-SDS running buffer. The precast gels contain no sodium dodecyl sulfate (SDS) and can therefore be used for either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or native gel electrophoresis depending upon the sample buffer and the running buffer used. Advantages of Mini-PROTEAN TGX precast gels: Increased stability and long shelf life up to 12 months Laemmli-like separation pattern Exceptionally straight lanes and sharp bands No need for special, expensive buffers Superior staining quality No gel foot to remove prior to blotting Bottom open cassette that unlocks with four easy clicks The Mini-PROTEAN Tetra cell runs both hand cast gels and Mini-PROTEAN TGX precast gels interchangeably. The cell can run from one to four gels, and the mini tank is compatible with other Bio-Rad electrode modules for tank blotting. The Mini-PROTEAN 3 cell runs both hand-cast and Mini-PROTEAN TGX precast gels. The cell can run one or two gels, and the mini tank is compatible with other Bio-Rad electrode modules for tank blotting, 2-D electrophoresis and electroelution. The Mini-PROTEAN 3 Dodeca cell is a multi-cell for high through-put system gel electrophoresis. It can run up to 12 identical polyacrylamide gels simultaneously. The Dodeca cell includes clamping frames, buffer dams, and a drain line. 1

1.2 Mini-PROTEAN Precast Gel Specifications Gel material Polyacrylamide Gel dimensions 7.2 x 8.6 cm (H x W) Gel thickness 1.0 mm Resolving gel height 6.2 cm Cassette dimensions 8.5 x 10 cm (H x W) Cassette material Styrene copolymer Comb material Polycarbonate Total running buffer volume 700 ml for 2 gels, 1,000 ml for 4 gels (Mini-PROTEAN Tetra Cell & Mini-PROTEAN 3) Storage conditions Store flat between 2 C and 8 C; DO NOT FREEZE Mini-PROTEAN Tetra Cell Specifications Casting stand Pin, retaining ring and spring Casting frames Gray gaskets Electrode assembly Electrodes Gasket, electrode inner core Tank and lid Sample loading guides Combs Mini-PROTEAN 3 Cell Specifications Electrode assembly Electrodes Gasket, electrode inner core Tank and lid Sample loading guides Combs Polycarbonate Stainless steel Polysulfone Thermoplastic rubber (gray) Glass filled polybutylene terephthalate Platinum wire, 0.010 diameter Silicone rubber (green) Polycarbonate Delrin Polycarbonate Glass-filled liquid crystal polymer Platinum wire, 0.010 diameter Silicone rubber (green) Molded polycarbonate Delrin Polycarbonate Mini-PROTEAN 3 Dodeca Cell Specifications Tank and lid Acrylic Clamping frame Polycarbonate and liquid crystal polymer Upper electrode holder Polycarbonate with 109 mm (4.3 ) platinum wire Lower electrode assembly Polycarbonate with 89 mm (3.5 ) platinum wire Drain line Tygon tubing Drain line connectors Delrin Cooling coil Acrylic Cooling coil connector tubing Tygon Maximum buffer volume 4.4 L Minimum buffer volume 3.4 L Overall size 41.5 x 15 x 16.2 cm (L x W x H) Safety limits 300 V, 150 W Weight 5 kg (11 lb) 2

1.3 Important Notes (See Appendix C for Related Literature) Mini-PROTEAN Tetra and Dodeca cell components are not compatible with acetone or ethanol. Use of organic solvents voids all warranties. Each Mini-PROTEAN precast gel should be used shortly after it is removed from the storage pouch. It is not advisable to run more than one gel type in the same apparatus at the same time. The different gel percentages will have different conductivity and therefore different run rates. When running 1 or 2 gels in the Tetra cell, use the electrode assembly (with the banana plugs), not the companion running module (without the banana plugs). When running 3 or 4 gels, both the electrode assembly and the companion running module must be used. When running 1 or 2 gels only, DO NOT place the companion running module in the tank. Doing so will cause excessive heat generation and degrade the quality of the electrophoretic separation. Improper storage of Mini-PROTEAN precast gels can produce numerous artifacts. Gels should be stored flat between 2 C and 8 C. Avoid freezing or prolonged storage above 8 C. If you suspect your gels have been stored improperly, THEY SHOULD BE DISCARDED. Do not attempt to lock the green arms of the electrode assembly without first ensuring that the gel cassettes are correctly aligned against the notches on the green gaskets of the module. To prevent the gels from shifting during the locking step, firmly and evenly grip them in place against the core of the module (see Figure 2c and 2e). 1.4 Mini-PROTEAN Comb Configurations Mini-PROTEAN TGX Gel Comb type Well volume 10 well 30 µl 15 well 15 µl IPG 7 cm ReadyStrip IPG strip Section 2 Setup and Basic Operation 2.1 Required Materials Clean Mini-PROTEAN Tetra cell tank Electrophoresis module: to run 1 or 2 gels, use the electrode module. To run 3 or 4 gels, use the electrode module and companion module PowerPac power supply or equivalent Sample buffer Running buffer (700 ml for 2 gels; 1,000 ml for 4 gels) Mini-PROTEAN precast gels 3

Fig. 1. Mini-PROTEAN Precast Gel Cassette. 2.2 Mini-PROTEAN Precast Gel Set Up Overview 1. Remove Comb: Position both thumbs on the ridges of the comb. Remove the comb by pushing upward in one smooth continuous motion. 2. Remove Tape: Pull gently to remove the green tape from the bottom of the cassette. 3. Rinse Wells: Use a syringe wash bottle or a disposable transfer pipette to rinse the wells with running buffer. Straighten the sides of the wells, if necessary. 4. Run Gel: Assemble the cassette into the running module of the Mini-PROTEAN system. Add running buffer to the inner and outer chambers. Prepare the samples in sample buffer and load the samples into the wells. Run the gel at 200 V until the dye front reaches the line on the bottom of the gel cassette (approximately 30 40 min). At the completion of the run, disconnect the cell and remove the cassette. 5. Open Cassette: Align the arrow on the opening key with the arrows marked on the cassette. Insert the key between the cassette plates at all 4 locations and apply downward pressure to break each seal. Do not twist the lever. Gently pull apart the two plates beginning from the top of the cassette. 6. Remove Gel: Gently remove the gel from the cassette. 4

2a 2b 2c Clamping Frame Gasket Notch 2d Gel Cassette Short Plate Long Plate 2f 2e Gel Support Fig. 2. Assembling the Mini-PROTEAN Tetra Cell Electrphoresis Module. 2.3 Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module 1. Set the electrode assembly to the open position on a clean flat surface (see Figure 2a) 2. Place the first gel cassette (with the short plate facing inward) onto the gel supports; gel supports are molded into the bottom of the electrode assembly. There are two supports on each side of the electrode assembly. Note that the gel will now rest at a 30 angle, tilting away from the center of the electrode assembly. Use caution when placing the first gel, making sure that the electrode assembly remains balanced and does not tip over. Place the second gel or buffer dam on the other side of the electrode assembly, again by resting the gel on the supports. At this point there will be two gels resting at a 30 angle, one on either side of the electrode assembly, tilting away from the center of the frame (see Figure 2b). It is critical that gel cassettes be placed into the electrode assembly with the short plate facing inward to form the inner buffer chamber. The electrode assembly requires two gels to create a functioning assembly; if an odd number of gels (1 or 3) is being run, you must use the buffer dam to complete the assembly (see Figure 2b). 3. Using one hand, gently push both gels toward each other, making sure that they rest firmly and squarely against the green gasket that is built into the electrode assembly. Align the short plates to ensure the edge sits just below the notch at the top of the green gasket (Figure 2e). 4. While gently squeezing the gel cassettes or a gel cassette and a buffer dam against the green gaskets with one hand (keeping constant pressure and both gels firmly held in place), slide the green arms of the clamping frame over the gels, locking them into place (see Figure 2c). 5

5. The wing clamps of the electrode assembly lift each gel cassette up against the notch in the green gasket, forming a seal (Figure 2d). Check again to make certain that the short plates sit just below the notch at the top of the green gasket (Figure 2e). Place the assembled electrophoresis module into the tank (Figure 2f) and fill the buffer chambers. At this point, the sample wells can be washed out with running buffer, if this was not done earlier, and the sample can be loaded. If running more than 2 gels, repeat steps 2a 2d with the companion running module. Section 3 SDS-PAGE 3.1 Introduction Mini-PROTEAN TGX precast gels provide a versatile system for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a gel electrophoretic technique that separates proteins according to their molecular weight. SDS-PAGE relies on a discontinuous buffer system. Two ions of differing electrophoretic mobility (glycinate and chloride) form a moving boundary when voltage is applied. Proteins have an intermediate mobility, causing them to concentrate, or stack, into a narrow zone at the beginning of electrophoresis. As the boundary moves through the gel, the sieving effect of the polyacrylamide gel matrix causes different proteins to move at different rates. The stacking effect is responsible for the high resolving power of SDS-PAGE. The sample is loaded in a relatively broad zone, and the moving boundary concentrates the proteins into sharp bands prior to separation. Protein samples for SDS-PAGE are prepared using SDS and a thiol reductant, usually 2-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins giving them a rod like shape and similar charge to mass ratio. The reductant cleaves disulfide bonds between and within proteins allowing complete denaturation and dissociation. Heat treatment in the presence of SDS and reductant effectively eliminates the effects of 2 and 3 protein structure and native charge on electrophoretic mobility, so the migration distance depends primarily on molecular weight. Molecular mass is determined by plotting the logarithm of protein molecular mass vs. the relative mobility (R f ) of the protein (R f = distance migrated by protein/distance relative to the dye front of the protein). Refer to tech notes 3133 and 3144. Mini-PROTEAN TGX precast gels are prepared without SDS. Although gels for SDS-PAGE have historically been cast with SDS in the gel, high quality SDS-PAGE separations are obtained in gels lacking SDS, provided that the sample buffer and running buffer contain sufficient SDS to maintain SDS saturation during electrophoresis. The recommended concentrations of SDS are >1% in the sample buffer and 0.1% in the running buffer. The absence of SDS in the gel itself provides additional flexibility, as the gels may also be used for native electrophoresis (see Section 4). Mini-PROTEAN TGX precast gels differ from the standard Laemmli system (Tris-HCl SDS-PAGE) gels due to a proprietary modification to their formulations that provides the gels with extended shelf life and improved separation characteristics. They are designed to be run using standard Laemmli sample and running buffers. No additional special buffers or reagents are required. 6

3.2 Mini-PROTEAN TGX Precast Gel Composition Mini-PROTEAN TGX gels are comprised of polyacrylamide with a bisacrylamide cross linker. Each gel has a 4% polyacrylamide stacking layer extending approximately 5 mm from the bottom of the loading well to the top of the resolving gel. The proprietary gel formulation provides a shelf life of 12 months and improved separation characteristics. The gel is packaged with storage buffer of the same composition with additional 0.02% sodium azide as a preservative. 3.3 Mini-PROTEAN TGX Precast Gel Selection Guide Mini-PROTEAN TGX gels are available in a wide selection of single percentages and gradients for the separation of proteins by SDS-PAGE. Gel Selection Guide Gel % Optimal Sample Running Run Conditions* Run Type Separation Range Buffer Buffer Voltage/Current** Time*** TGX 7.5 40 200 kd SDS-PAGE SDS-PAGE 200 V constant 38 min sample buffer running Starting current buffer (per gel): 37 ma Final current (per gel): 23 ma TGX 10 30 150 kd SDS-PAGE SDS-PAGE 200 V constant 38 min sample running Starting current buffer buffer (per gel): 37 ma Final current (per gel): 23 ma TGX 12 20 120 kd SDS-PAGE SDS-PAGE 200 V constant 38 min sample running Starting current buffer buffer (per gel): 37 ma Final current (per gel): 23 ma TGX 4 15 20 250 kd SDS-PAGE SDS-PAGE 200 V constant 30 min sample running Starting current buffer buffer (per gel): 50 ma Final current (per gel): 33 ma TGX 4 20 10 200 kd SDS-PAGE SDS-PAGE 200 V constant 30 min sample running Starting current buffer buffer (per gel): 50 ma Final current (per gel): 33 ma TGX Any kd****10 200 kd SDS-PAGE SDS-PAGE 200 V constant 28 min sample running Starting current buffer buffer (per gel): 50 ma Final current (per gel): 33 ma *This may vary depending on water and buffer conductivity, which may vary from one lab setting to the next. **Current should be multiplied by the number of gels being run. ***Approximate time required for dye front to reach the line at the bottom of the cassette. ****Any kd is a unique single percentage formulation that provides a broad separation range and short running time. 7

3.4 SDS-PAGE Buffers See Section 5 for buffer recipes. 3.5 Sample Preparation The appropriate concentration of sample depends on the load volume and the detection method used. (See Section 6 for approximate stain sensitivities). Add 50 µl 2-mercaptoethanol per 950 µl of sample buffer for a final concentration of 5% 2-mercaptoethanol, or 710 mm. As an alternative, DTT may be used at a final concentration of 350 mm (54 mg/ml). Dilute 1 part sample with at least 1 part sample buffer with added reductant. Heat the mixture at 95 C for 5 min. 3.6 Running Conditions Run gels at 200 V constant voltage until the dye front reaches the line near the bottom edge of the gel cassette. Approximate run times will vary between 28 and 38 min depending on the gel type (see Section 3.3). Section 4 Native PAGE 4.1 Introduction Mini-PROTEAN TGX gels are made without SDS, allowing native separations using SDS- and reductant-free sample and running buffers. The nonreducing and nondenaturing environment of native PAGE allows protein separation with retention of biological activity. Native PAGE can also be used to resolve multiple protein bands when molecular mass separation by SDS-PAGE would reveal only one. Native PAGE uses the same moving boundary described in Section 3.1. Proteins are prepared in nonreducing nondenaturing sample buffer, which maintains the proteins secondary structure and native charge density. Protein mobility depends on the size and shape of the protein as well as its molecular weight and net charge. Native PAGE is therefore not suitable for molecular weight determination. 4.2 Native PAGE Buffers See Section 5 for buffer recipes. 4.3 Sample Preparation Determine the desired protein concentration and load volume of your sample based on the detection method used. (See Section 6 for approximate stain sensitivities). Proteins can be separated using a standard protocol, following dilution of the sample with an equal volume of Native Sample Buffer (see Section 5, DO NOT HEAT SAMPLES). Strongly basic proteins (pl >8.5) will have a net positive charge and will not enter a native PAGE TGX gel. 4.4 Running Conditions See Section 3.3. 8

Section 5 Buffers (see Appendix A for Stock Solutions) Name Working Concentration Notes Pre-Mixed Alternative SDS-Page running buffer 1X 25 mm Tris-base 192 mm glycine 0.1% (w/v) SDS Running buffer should be ~ ph 8.3. Do not adjust the ph 10x Tris/Glycine/SDS, 1 L, 161-0732 10x Tris/Glycine/SDS, 5 L cube, 161-0772 SDS-PAGE sample buffer Native PAGE running buffer working concentration Native PAGE sample buffer 2X 62.5 mm Tris-HCl, ph 6.8 2% (w/v) SDS 25% (v/v) glycerol 0.01% (w/v) Bromophenol Blue 5% (v/v) 2-mercaptoethanol or 350 mm DTT (added fresh) 25 mm Tris -Base 192 mm glycine 62.5 mm Tris-HCl, ph 6.8 40% glycerol 0.01% Bromophenol Blue Dilute 1 part sample with 1 part sample buffer. More sample buffer can be added if necessary. 1 part sample to 2 parts sample buffer dilution also works. Dry samples can be dissolved directly into the sample buffer Running buffer should be ~ ph 8.3. Do not adjust the ph Dilute 1 part sample with 1 part sample buffer. More sample buffer can be added if necessary. 1 part sample to 2 parts sample buffer dilution also works. Dry samples can be dissolved directly into the sample buffer Laemmli sample buffer, 30 ml, 161-0737 10x Tris/Glycine, 1 L, 161-0734 10x Tris/Glycine, 5 L cube, 161-0771 Native sample buffer, 30 ml, 161-0738 9

Section 6 Total Protein Gel Stains for SDS-PAGE and Native PAGE Detection Method Sensitivity Optimal Protein Load Coomassie Blue R-250 Bio-Safe Coomassie stain 36 47 ng ~0.5 8 28 ng ~0.5 Zinc stain 6 12 ng ~0.2 Silver Stain Plus kit 0.6 1.2 ng ~0.01 Silver stain 0.6 1.2 ng ~0.01 Dodeca Silver Stain Kit SYPRO Ruby protein gel stain Flamingo Fluorescent Oriole Fluorescent protein gel stain 0.5 1.2 ng ~0.1 1 10 ng ~0.2 0.25 0.5 ng ~0.01 0.5 ng ~0.2 Advantages Disadvantages Imaging Instruction Manual Number Laboratory standard Nonhazardous High-contrast, fast, reversible stain Simple, robust, mass spectrometry compatible Stains complex proteins, i.e., glycoproteins, and lipoproteins Convenient staining for a large number of gels Broad dynamic range, simple robust protocol Broad dynamic range, mass spec compatible High sensitivity Broad dynamic range Requires MeOH Negative stain, must be photographed, SDS-PAGE only Does not stain Glycoproteins well Not mass spectrometry compatible Requires imaging instrument for maximum sensitivity Requires imaging instrument for maximum sensitivity Will not work with visible excitation Photography with white light or transmission (Gel Doc or GS-800 ) Photography with white light or transmission (Gel Doc or GS-800) Photography with white light or transmission (Gel Doc or GS- 800) Photography with white light or transmission (Gel Doc or GS-800) Photography with white light or transmission (Gel Doc or GS-800) Photography with white light or transmission (Gel Doc or GS- 800) Fluorescence visualization with UV transillumination or laser scanning Fluorescence visualization with UV transillumination or laser scanning (best option) Fluorescence visualization with UV transillumination (Gel Doc, Chemi Doc) Consult scientific literature 4307051 4006082 LIT442 LIT34 4110150 4006173 10003321 10017295 10

Section 7 Troubleshooting Problem Cause Solution Current is zero or less than Tape at the bottom of Remove tape expected and samples do not the cassette not migrate into gel removed Insufficient buffer in inner Fill buffer chamber with buffer chamber 700 ml running buffer Insufficient buffer Make sure the inner in outer buffer and outer buffer chamber chambers are sufficiently filled to ensure that the wells of the gel are completely covered Electrical disconnection Check electrodes and connections Bands smile across gel, Excess heating of gel Check buffer composition band pattern curves upward Do not exceed at both sides of the gel recommended running conditions Insufficient buffer Make sure the inner and outer buffer chambers are sufficiently filled to ensure that the wells of the gel are completely covered Smiling or frowning bands Overloaded proteins Load less protein within the gel lane Sample Consider minimizing preparation/buffer salts, detergents and issues solvents in sample preparation and sample buffer Running speed Check to make sure the correct voltage has been set Skewed or distorted bands, Excess salt in samples Remove salts from lateral band spreading sample by dialysis or desalting column prior to sample preparation Insufficient sample Check buffer buffer or wrong composition and formulation dilution instructions Vertical streaking Overloaded samples Dilute sample 11

Problem Cause Solution Vertical streaking Sample precipitation Selectively remove predominant protein in the sample Dilute sample in more sample buffer Insoluble materials Centrifuge samples to in the samples remove particulates (e.g., membranes) prior to sample loading Gels run faster than Running buffer is too Check buffer expected concentrated and gel composition temperature is too high Incorrect running buffer type is used Artifact bands at ~60 70 kd Possible skin keratin Thoroughly clean all contamination dishware and wear gloves while handling and loading gel Filter all solutions through 0.2 µm or 0.45 µm filter Leaking from inner Incomplete gasket seal Wet the gasket with buffer chamber running buffer before use Improper assembly of Check that the top edge the gel into the of the short plate fits electrode/companion under the notch at the assembly top of the gasket Make sure that the top of the short plate is touching the green gasket Poor resolution High sample volume If possible, load a more or fuzzy bands concentrated sample in a lower volume of sample buffer Diffuse sample loading Load sample with zone syringe or gel loading pipette tips Sample diffusion during Fix gel with 40% staining with Coomassie methanol, 10% acetic stain acid for 80 min prior to staining Incompatible sample Consider minimizing components salts, detergents, and solvents in sample preparation and sample buffer Bands are not present Proteins have Use a smaller pore or are missing from the transferred through the size membrane blotting membrane* membrane Decrease the transfer time Decrease the voltage *For more Western blot troubleshooting suggestions, see the Mini Trans-blot Electrophoretic Transfer Cell Instruction Manual (1703930) or the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell Instruction Manual (1703940). 12

Appendix A Stock Solutions Buffer Notes SDS-PAGE running 10x Stock Running buffer should buffer Tris base 15.0 g be ~ph 8.3. Do not adjust Glycine 72.0 g the ph SDS 5.0 g To 500 ml with DI H 2 O SDS-PAGE sample 2x Stock buffer 0.5M Tris-HCl, ph 6.8 1.0 ml 10% (w/v) SDS 1.6 ml Glycerol 2.0 ml 1.0% Bromophenol Blue 0.08 ml 2-Mercaptoethanol 0.4 ml DI H 2 O 2.92 ml Total Volume 8.0 ml Native PAGE running 10x Stock Running buffer should buffer Tris base 15.0 g be ~ph 8.3. Do not adjust Glycine 72.0 g the ph To 500 ml with DI H 2 O Native PAGE sample 2x Stock buffer 0.5M Tris-HCl, ph 6.8 1.0 ml Glycerol 2.2 ml 1% Bromophenol Blue 0.08 ml DI H2O 3.72 ml Total Volume 8.0 ml 0.5 M Tris-HCl Tris base 6.06 g Adjust to ph 6.8 with HCl. DI H 2 O ~60 ml Make to 100 ml with Total Volume 100 ml DI H 2 O. Store at 4 C 10% SDS SDS 1.0 g Stir gently To 10 ml with DI H 2 O 1% Bromophenol Blue Bromophenol Blue 100 mg Stir gently To 10 ml with DI H 2 O Coomassie Blue R-250 Methanol (40%) 400 ml Dissolve Coomassie R-250 staining solution (0.1%) Acetic Acid (10%) 100 ml in methanol/acetic acid. Coomassie Blue R-250 (0.1%) 1.0 g Add DI H 2 O to a final To 1,000 ml with DI H 2 O volume of 1,000 ml Coomassie Blue R-250 Methanol 400 ml destaining solution Acetic acid 100 ml DI H 2 O 500 ml 13

Appendix B Total Protein Blot Stains Method Sensitivity Optimal Protein Load SYPRO Ruby protein blot stain Colloidal Gold stain Amido Black 10B 2 8 ng ~0.2 1 ng ~0.1 Advantages Disadvantages Imaging Compatible with mass spectrometry, Edman-based sequencing, and standard immunological procedures Sensitive, one step 100 1,000 ng ~5.0 Standard membrane, economical Multiple step protocol; requires imaging instrument for maximum sensitivity Not compatible with nylon membranes Low sensitivity stain Fluorescence visualization with UV epiillumination or laser scanning Photography with white light or reflectance Photography with white light or reflectance Instruction Manual Number 4006173 LIT294 9130 Appendix C Related Literature Name Bulletin Number Mini-PROTEAN Tetra Cell Instruction Manual 10007296 Mini-PROTEAN 3 Instruction Manual 4006157 Mini-PROTEAN 3 Dodeca Cell Instruction Manual 4006191 Mini Trans-Blot Instruction Manual M1703930 Criterion Blotter Instruction Manual 4006190 Trans-Blot Cell Instruction Manual 1703910 Trans-Blot SD Cell Quick Reference Guide 4006066 Trans-Blot SD Semi-Dry Transfer Cell Instruction Manual 1703940 Blotting Membrane Brochure 1939 Western Blotting Detection Reagent Brochure 2032 Ready-to-Run Buffers and Solutions Brochure 2317 Little Book of Standards 2414 Model 583 Gel Dryer Instruction Manual M1651740 GelAir Drying System Instruction Manual 4006040 14

Appendix D Ordering Information D.1 Mini-PROTEAN TGX Precast Gels 10 Gels per box 2 Gels per box 10-Well 15-Well IPG Comb 10-Well 30 µl/well 15 µl/well 7 cm IPG Strip 30 µl 7.5% 456-1023 456-1026 456-1021 456-1023S 10% 456-1033 456-1036 456-1031 456-1033S 12% 456-1043 456-1046 456-1041 456-1043S 4 15% 456-1083 456-1086 456-1081 456-1083S 4 20% 456-1093 456-1096 456-1091 456-1093S Any kd 456-9033 456-9036 456-9031 456-9033S D.2 Premixed Running and Sample Buffers Catalog Number Product Description 161-0732 10x Tris/Glycine/SDS, 1 L 161-0772 10x Tris/Glycine/SDS, 5 L cube 161-0737 Laemmli Sample Buffer, 30 ml 161-0738 Native Sample Buffer, 30 ml 161-0734 10x Tris/Glycine, 1 L 161-0771 10x Tris/Glycine, 5 L cube 161-0778 10x Tris/CAPS, 1 L 161-0780 10x Phosphate Buffered Saline, 1 L 170-6435 10x Tris Buffered Saline, 1 L 161-0783 1x Phosphate Buffered Saline With 1% Casein, 1 L 161-0782 1x Tris Buffered Saline With 1% Casein, 1 L D.3 Individual Reagents Catalog Number Product Description 161-0719 Tris, 1 kg 161-0716 Tris, 500 g 161-0717 Glycine, 250 g 161-0718 Glycine, 1 kg 161-0724 Glycine, 2 kg 161-0301 SDS, 100 g 161-0302 SDS, 1 kg 161-0416 SDS Solution, 10% (w/v), 250 ml 161-0418 SDS Solution, 20% (w/v), 1 L 170-6404 Blotting-Grade Blocker, 300 g 161-0710 2-Mercaptoethanol, 25 ml 161-0610 Dithiothreitol, 1 g 161-0611 Dithiothreitol, 5 g 161-0404 Bromophenol Blue, 10 g 15

D.4 Total Protein Gel and Blot Stains Catalog Number Product Description 161-0786 Bio-Safe Coomassie Stain, 1 L 161-0400 Coomassie Brilliant Blue R-250, 10 g 161-0436 Coomassie Blue R-250 Stain Solution, 1 L 161-0438 Coomassie Blue R-250 Destain Solution, 1 L 161-0443 Silver Stain Kit 161-0449 Silver Stain Plus Kit 161-0481 Dodeca Silver Stain Kit 170-6527 Colloidal Gold Total Protein Stain, 500 ml 161-0440 Zinc Stain and Destain Kit 170-3127 SYPRO Ruby Protein Blot Stain, 200 ml 170-3125 SYPRO Ruby Protein Gel Stain, 1 L 161-0490 Flamingo Fluorescent Gel Stain (10 x), 20 ml 161-0491 Flamingo Fluorescent Gel Stain (10 x), 100 ml 161-0492 Flamingo Fluorescent Gel Stain (10 x), 500 ml 161-0495 Oriole Fluorescent Protein Gel Stain (1 x), 200 ml 161-0496 Oriole Fluorescent Protein Gel Stain (1 x), 1 L 161-0497 Oriole Fluorescent Protein Gel Stain Kit, 5 L 16

D.5 Immunoblot Detection Method Sensitivity Optimal Advantages Disadvantages Imaging Protein Load 4CN colorimetric (HRP) 500 pg ~0.25 Fast detection Results may fade Photography with white light or reflectance DAB colorimetric (HRP) Opti-4CN colorimetric (HRP) Amplified Opti- 4CN colorimetric (HRP) 500 pg ~0.25 100 pg ~0.05 10 pg ~0.005 Fast detection Color does not fade High sensitivity, low background Contains toxic chemicals More expensive than 4CN Amplification requires additional steps Photography with white light or reflectance Photography with white light or reflectance Photography with white light or reflectance BCIP/NBT colorimetric 100 pg ~0.05 Sensitive, multiple antigen May detect endogenous (AP) enzyme activity Photography with white light or reflectance Immun-Star chemiluminescent (AP) 10 pg ~0.005 Long-lasting signal, short and multiple exposures possible Requires visualization on film or instrumentation Chemiluminescent visualization with film or imager Immun-Star chemiluminescent HRP 1 3 pg ~0.005 Intensifies signal output, very sensitive Requires visualization on film or instrumentation Chemiluminescent visualization with film or imager Immun-Star WesternC (HRP) 10 fg ~0.005 Long-lasting signal, short and multiple exposures possible Requires visualzation on film or instrumentation Chemiluminescent visualization with film or imager See related literature in Appendix C for information on Western blotting and gel drying. D.6 Immunoblot Detection Reagents Catalog Number Product Description 170-5070 Immun-Star WesternC Chemiluminescent Kit, 100 ml 170-6431 HRP Conjugate Substrate Kit, 4CN 170-6535 HRP Color Development Reagent, DAB, 5 g 170-8238 Amplified Opti-4CN Substrate Kit 170-8235 Opti-4CN Substrate Kit 170-6432 AP Conjugate Substrate Kit 170-5012 Immun-Star Substrate Pack 17

D.7 Blotting Membranes Catalog Number Product Description 162-0232 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack 162-0233 0.2 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack 162-0234 0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack 162-0235 0.45 µm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack 162-0236 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack 162-0237 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack D.8 Protein Standards Catalog Number Product Description 161-0363 Precision Plus Protein Unstained Standards (10 250 kd), 1,500 µl, 150 applications 161-0373 Precision Plus Protein All Blue Prestained Standards (10 250 kd), 500 µl, 50 applications 161-0374 Precision Plus Protein Dual Color Standards (10 250kD), 500 µl, 50 applications 161-0375 Precision Plus Protein Kaleidoscope Standards (10 250 kd), 500 µl, 50 applications 161-0376 Precision Plus Protein WesternC Standards (10 250kD), 250 µl, 50 applications 161-0385 Precision Plus Protein WesternC Pack (10 250kD), 50 applications each of standard and StrepTactin-HRP 161-0317 SDS-PAGE Standards, broad range, 200 µl 161-0320 2-D SDS-PAGE Standards, Unstained, 500 µl D.9 Equipment Catalog Number Product Description 165-8004 Mini-PROTEAN Tetra Cell 165-4100 Mini-PROTEAN 3 Dodeca Cell 170-3930 Mini Trans-Blot Electrophoretic Transfer Cell 170-3940 Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell 164-5050 PowerPac Basic Power Supply 164-5052 PowerPac HC High-Current Power Supply 164-5070 PowerPac Universal Power Supply 164-5056 PowerPac HV Power Supply 165-1789 Hydrotech Gel Drying System, 100/120V 165-1790 Hydrotech Gel Drying System, 220/240V 165-1771 GelAir Drying System, 115V, 60Hz 165-1772 GelAir Drying System, 230V, 50Hz SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad is licensed to sell SYPRO products for research use only, under US Patent 5,616,502. 18

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