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Copper staining31 (above) is advisable for the visualization of the bands in preparative SDS-PAGE, since this method does not employ fixative solvents. Desired bands are cut from the gel and destained by incubation in three changes (for 10 min each) of 0.25 M EDTA, 0.25 M Tris-Cl, pH 9. After destaining, gel slices are incubated in the appropriate elution buffer. Proteins are often extracted from macerated gel slices by simple diffusion into appropriate buffers or by solubilization of the gel. 5,33In the latter method, cross-linkers other than bisacrylamide are copolymerized into the gels. 5,7 For example, gels cross-linked with N,N'-bisacrylylcystamine (BAC) are dissolvable in 2-mercaptoethanol or dithiothreitol, while both N,N'-dihydroxyethylenebisacrylamide (DHEBA) and N,N'-diallyltartardiamide (DATD) result in gels which can be solubilized with periodic acid. Once gels have been dissolved, proteins must be separated from the large excess of gel material by gel filtration or ion-exchange chromatography. Electrophoretic elution is an efficient method for recovering proteins from gel slices. 2,5,8 In the simplest versions of this method, proteins are electrophoresed out of gel pieces into dialysis sacks in the types of apparatus used for running cylindrical gel rods. Devices are available for the rapid recovery of proteins in small volumes with yields of greater than 70% in most cases. Elution takes about 3 hr at 10 mA/tube in 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3 (standard SDS-PAGE electrode buffer). SDS can be removed from the eluted samples by dialysis or ionexchange chromatography)4 34A. J. Furth, Anal. Biochem. 109, 207 (1980).
[34] P r o t e i n Analysis U s i n g H i g h - R e s o l u t i o n T w o - D i m e n s i o n a l P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s By BONNIE S. DUNBAR, HITOMI KIMURA, and THERESE M. TIMMONS
The term two-dimensional electrophoresis has been used to describe a variety of methods employing separation of molecules in two dimensions. The term high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now specifically applied to the separation of proteins in the first dimension according to their isoelectric points using isoelectric focusing (IEF) with carrier ampholytes after reduction of disulfide bonds, METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
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followed by separation in the second dimension according to their molecular weights using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as defined by O'Farrell. 1 The history of the major developments in the field of electrophoresis and 2D-PAGE has been summarized in detail elsewhere. 2-4 The most significant recent advances in this technology have come through the standardization of equipment and reagents and the simplification of equipment for reproducible analysis and large scale analyses. 3,5 Because large numbers of laboratories are now using these standardized procedures, they are the methods described in this chapter. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. The use of 2D-PAGE has become increasingly popular during the past decade. Two-dimensional PAGE allows the resolution of a complex protein mixture into more discrete components than 1D-PAGE since it separates on the basis of protein charge in addition to molecular weight. The major advantage of large-scale 2D-PAGE is the improvement in reproducibility of protein patterns. This enables the researcher to directly compare the analyses of complex protein mixtures, whether the 2D-PAGE separations are conducted simultaneously or in different experiments. This feature makes 2D-PAGE a versatile and powerful tool in both basic and clinical research. Applications of 2D-PAGE The most common uses of 2D-PAGE are the analysis of complex mixtures of proteins and the analysis of the posttranslational modification of proteins. 2D-PAGE can also provide valuable information about the molecular properties of proteins, including an estimate of the relative isoelectric points (pI) and molecular weights 3 of proteins. However, it is generally inadequate to use this as the sole method for the precise determination of these parameters. For example, the disulfide bonds of the i p. H. O'Farrell and J. I. Garrels, this series, Vol. 100, p. 411. 2 N. G. Anderson and L. Anderson, Clin. Chem. 28, 739 (1982). 3 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. 4 B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C., 1981. L. Anderson, "Two-Dimensional Electrophoresis: Operation of the ISO-DALT System." Large Scale Biology Press, Washington, D.C., 1988.
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proteins analyzed by 2D-PAGE are usually reduced so the protein patterns may reflect subunit peptides. The pI and molecular weight values observed may therefore be different from those of the native proteins. One should be careful not to overinterpret data obtained from electrofocusing and 2D-PAGE. Another common use of 2D-PAGE is to rapidly purify a specific protein which can be cut from the gel and used directly to obtain amino acid sequence or to purify antibodies. These antibodies can then be used to immunoaffinity purify the original protein in quantities sufficient for detailed chemical characterization. Immunoblotting using antibodies to detect antigens separated by 2DPAGE also provides an excellent method to analyze antibody specificity and to analyze carbohydrate or other epitopes. Finally, the use of 2D-PAGE with silver staining provides one of the best methods to estimate protein purity. This analysis, in conjunction with one-dimensional analysis of proteins visualized by silver stain (to detect proteins whose pI is outside the pH range of the ampholines), will provide a rigorous estimate of protein purity. Sample Preparation and Solubilization Procedures The preparation of samples for 2D-PAGE analysis is the most critical step in guaranteeing excellent reproducible results. All tissues and samples should be handled in the cold and stored at -70 °. It is important that the ratio of solubilization buffer to protein concentration be optimized for each sample. We have found the following ratios to be adequate for most samples: (1) 200-500/zg tissue homogenate/2 ml solubilization buffer, (2) 20-50/xl cell pellet/300/zl solubilization buffer, (3) 1 x 10 6 cells in tissue culture plate with 500/zl solubilization buffer to solubilize cells directly, and (4) 10-200/xg soluble protein/30-50/zl solubilization buffer. Note: 550/zl of each of the above samples should be adequate for identification of abundant proteins by Coomassie Blue staining or of minor proteins by silver staining in two-dimensional gels. Materials
Sodium dodecyl sulfate (SDS) (Bio-Rad, Richmond, CA) Cyclohexylaminoethane (CHES) (Calbiochem, San Diego, CA) Glycerol (Fisher, Pittsburgh, PA) 2-Mercaptoethanol (Bio-Rad) Urea (ultrapure) (Bio-Rad) Nonidet P-40 (nonionic detergent, NP-40) (Accurate Chemical, Westbury, NY)
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Ampholytes [pH 3.5-10: Bio-Rad, LKB, or Pharmacia (Piscataway, NJ): pH 2-11: Serva, Garden City Park, NY]. This wide-range mixture is appropriate for most routine samples. Other pH range or combinations of brands of ampholytes may be used in some instances H20, deionized with mixed bed resin (Continental filter system) or deionized double-distilled H20 Method
The two solubilization buffers which can be used for isoelectric focusing are as follow: SDS solubilization solution: 0.05 M CHES, 2% SDS, 10% glycerol, small amount of Bromphenol Blue, pH 9.5. Add 2% 2-mercaptoethanol just before use. Samples should be suspended in SDS solubilization buffer, placed in a tightly capped glass vial, and heated for 510 min in a boiling water bath. (Thick plastic tubes such as microfuge tubes are insulated and interfere with heating.) It may be necessary to solubilize some samples at room temperature for 2-3 hr, with or without heating Urea solubilization solution: 9 M urea, 4% Nonidet P-40. Add 2% 2mercaptoethanol and 2% ampholytes to a small aliquot of solubilization buffer just prior to use. These reagents should be filtered to 0.2 /zm with a syringe filter for best results. Samples should be suspended in the urea solubilization solution and incubated at room temperature for 2 hr. Caution: Do not heat, or you will generate charge artifacts Following the incubation, samples are centrifuged to remove nonsolubilized material and nucleic acids that may interfere with focusing or cause streaking in second-dimension protein patterns (100,000-200,000 g for 2 hr is suggested). We recommend using a Beckman Ti-42.2 rotor, which holds 72 tubes. Isoelectric Focusing Materials
Urea (ultrapure) (Bio-Rad) Ampholytes (LKB, Serva, or Pharmacia recommended); pH will depend on needs of investigator Acrylamide (Bio-Rad) Bisacrylamide (Bio-Rad) Nonidet P-40 (Accurate) Ammonium persulfate (Bio-Rad)
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N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad) Sodium hydroxide (Sigma, St. Louis, MO) Phosphoric acid (Fisher) Chromerge (Fisher) Glass pipet tubes (0.2 ml disposable) (American Scientific Products, S/P disposable serological pipets, 0.2 ml, #P4644-2T) Gel electrophoresis apparatus: Any tube gel electrophoresis apparatus can be used if appropriate grommets or corks are prepared to fit small tubes (e.g., Bio-Rad electrophoresis unit model 175 tube gel apparatus). Alternatively, multiple IEF casting systems now available from Pierce Apparatus Branch, Hoefer Scientific, and Integrated Separation Sciences have been optimized for these procedures and are highly recommended Method To cast IEF gels, add urea (8.25 g) to 6 ml HzO plus 2.0 ml acrylamide stock (30 g acrylamide : 1.8 g bisacrylamide : 100 ml HzO, filtered to 0.2 tzm). Dissolve the urea in the H20 by swirling the flask under warm running water. Caution: Do not heat solution. Add ampholytes (0.75 ml) to the mixture of acrylamide, water, and urea, swirl the solution gently to mix, and degas on a lyophilizer. Add 0.3 ml Nonidet P-40, and mix gently. (Hint: A large, plastic Eppendorf pipet tip can be cut off for easier and more accurate pipetting of viscous detergents.) Add ammonium persulfate (70 txl of a 10% solution) and TEMED (10/zl) to the acrylamide solution, and swirl the flask gently to mix. Cast IEF gels to a height of approximately 12 cm using capillary action, by overlayering acrylamide stock with water using a commercial casting apparatus, or a home-made casting chamber prepared from 2- to 50-ml plastic conical centrifuge tubes (as described in Dunbar3). Allow 1 hr for polymerization, and place tubes into the electrophoresis chamber. Prepare upper electrode buffer (0.02 N NaOH degassed thoroughly on lyophilizer) and lower electrode buffer (0.085% phosphoric acid), and add to chamber. Prefocus the gels at 200 V for 1-2 hr. In theory, this will remove ions which may interfere with the focusing. We have frequently omitted this step, however, with no noticeable differences in protein patterns. Load the protein samples (5-50 ~1) with a Hamilton syringe under the upper electrode buffer. Carry out isoelectric focusing for 10,000-12,000 V-hr (e.g., 17 hr at 700 V). The optimal conditions will depend on the nature of your sample and the dimensions and volume of your IEF gels. We have found that resolution of proteins is sharper if you focus for a shorter period of time at higher voltage (i.e., 700 V for 16 hr is better than 500 V for 22 hr).
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To remove gel, insert a yellow Eppendorf pipet tip attached to a 3-ml syringe filled with water into the top of the tube, and gently push out the gel. The IEF gels are equilibrated in buffer (0.125 M Tris-base; 2% SDS; 10% glycerol, pH 6.8, plus 0.2-0.8% 2-mercaptoethanol added just before use) for 15 min, to remove ampholytes and urea and to recoat the proteins with SDS. In some instances, we have equilibrated the gels for as little as 2-5 min with excellent results. Note: The "mercaptan" artifact commonly observed by silver staining which appears as two distinct lines having molecular weights of approximately 50K and 70K can be reduced if little or no 2-mercaptoethanol is used in the equilibration buffer. You should first establish whether the omission of this reducing agent will alter your protein patterns by comparing samples run in its presence or absence. The IEF gel can be frozen at - 7 0 ° before equilibration, and thawed in equilibration buffer immediately before placing on the surface of the second dimension slab gel. Nonequilibrium pH Gradient Electrophoresis (NEPHGE Gel System) in 2D-PAGE "Nonequilibrium" isoelectric focusing techniques are especially useful for the first dimension separation of basic proteins, which are not well resolved or cannot be resolved by other IEF procedures. 6 Samples must be solubilized in the urea solubilization buffer above. All gel-casting procedures should be carried out as for standard equilibrium IEF, except that the upper and lower buffers are reversed: the upper electrode buffer should contain phosphoric acid, and the lower buffer should contain sodium hydroxide. When attaching the electrodes to the power supply, be sure to attach the upper buffer reservoir to the positive electrode and the lower buffer reservoir to the negative electrode. Finally, the IEF gels should be removed at intervals such as 2000, 4000, 6000, or 8000 V-hr. Total volt-hours will have to be optimized to resolve different proteins of interest, since this is a nonequilibrium system. Casting and Running Individual One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gels for Second Dimension Electrophoresis Materials
Acrylamide (Bio-Rad, Polysciences, Serva Fine Chemicals, or Sigma): Reagents from the latter two sources are less expensive, but require filtering through Whatman #3 filter paper, followed by a 0.2-~ Milli6 p. Z. O'Farrell, H. M. G o o d m a n , a n d P. H. O'Farrell, Cell 12, 1133 (1977).
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pore filter. These impure reagents may also have contaminants detectable by silver stain. They can easily be distinguished from protein spots in 2D-PAGE, but will make analysis of silver-stained 1D-PAGE difficult N,N'-Methylene(bis)acrylamide (Bio-Rad) Trizma base (Sigma) Glycine (Sigma) SDS (Bio-Rad) 2-Mercaptoethanol (Bio-Rad) Glycerol (Fisher) N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad) sec-Butanol (Fisher) Agarose (Bio-Rad) Glass plates and spacers for individual or multiple gel-casting systems: The size plates will depend on the type of electrophoresis chamber that will be utilized (e.g., 18 x 16 cm plates with 1.5-cm spacers are compatible with Bio-Rad or Hoefer electrophoresis units). The recipes in this chapter are for this size gel Electrophoresis chambers: These can be obtained commercially from Bethesda Research Laboratories, Bio-Rad, Hoefer, or can be custom made (Studier apparatus) Gradient maker (double conical style recommended) Multiple electrophoresis gradient gel-casting systems (highly recommended if you do gels regularly; greatly improves reproducibility!)
Method for Casting Nongradient Gels Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide (filter to 0.2 t~m) Gel buffer stock: 1.5 M Trizma base, 0.4% SDS (filter to 0.2/~m) Ammonium Persulfate: 10 g ammonium persulfate; final volume 100 ml (filter to 0.2 t~m). Freeze at - 2 0 ° in small aliquots to guarantee the consistency of polymerization for as long as the stock lasts Tank buffer: 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS Assemble the gel-casting apparatus. Combine acrylamide, buffer, and H20; degas (see tabulation below for final acrylamide concentration). Add TEMED and mix thoroughly but gently by swirling the beaker. Add ammonium persulfate and swirl gently to mix. Pour the mixture down one edge of the spacer of the gel-casting unit using a 25-ml pipet, or a syringe and a large (18-gauge) needle. Fill to within 3 cm of the top of the glass plates. Carefully overlay with water-saturated sec-butanol, and allow to
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polymerize for 45-60 min. (Note: This is the most important step for obtaining good resolution in slab polyacrylamide gels.) When the gel is polymerized, rinse the surface several times with distilled water, and drain well. Final acrylamide concentration Component
7.5%
10%
12.5%
15%
Bisacrylamide stock Gel buffer stock H20 10% ammonium persulfate TEMED
7.1 ml 7.1 ml 14.2 ml 105/.tl 15/zl
9.5 ml 7.1 ml 11.8 ml 105/~1 15/zl
11.8 ml 7. I ml 9.5 ml 105/~1 15/~1
14.2 ml 7.1 ml 7.1 ml 105/~1 15/zl
Method for Casting Gradient Gels If silver staining methods are to be used, all reagents should be filtered to 0.2/zm. Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide Second dimension buffer stock: 40 g Trizma base, 20 g Trizma-HCl; final volume 300 ml, pH 8.5-8.6 10% second dimension buffer: Three parts second dimension buffer stock plus five parts H 2 0 20% second dimension buffer: Three parts second dimension buffer stock plus one part glycerol 10% SDS: 10 g SDS; final volume 100 ml Assemble individual slab gel units in casting apparatus. For each gradient gel (approximately 40 ml/volume), prepare the reagents as per the following tabulation:
Gradient mix
Second dimension bisacrylamide stock (ml)
10% second dimension buffer (ml)
20% second dimension buffer (ml)
10% SDS (ml)
10% ammonium persulfate (gl)
10% 20%
6.3 14.7
14.7 0.0
0.0 6.3
0.2 0.2
30 15
TEMED (/xl) 2
2
Place 20% gradient mix in the internal .chamber of the gradient maker and begin mixing with a magnetic stir bar. Place 10% gradient mix in the
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external chamber, start the peristaltic pump, and open the gradient chamber to begin gradient formation. When the gradient is finished, spray the surface of the gel with water-saturated sec-butanol. After polymerization is complete, rinse the surface of the gel with HE0 and drain well.
Loading and Running Individual Second Dimension Polyacrylamide Gels Set the polymerized slab gel on a loading stand and lay the IEF gel on a platform (or sheet of parafilm) and gently straighten out. Allow the gel to slide into place along the surface, being sure not to trap air bubbles. If desired, seal the IEF gel with a small amount of overlay agarose (0.25 M Trizma base, 0.192 M glycine, 0.1% SDS, and 0.5% agarose heated to dissolve thoroughly, and then cooled slightly before overlayering gel). If standard electrophoresis chambers are used (e.g., Studier, Bio-Rad, or Hoefer electrophoresis apparatus), electrophoresis is carried out by placing the electrode buffer in the upper and lower chambers. The slab gels are then placed into these chambers, taking care to avoid air bubbles being trapped at the bottom of the slab acrylamide gel. This can be done by tilting the gel as it is lowered into the chamber and by tilting the chamber so that the buffer will move across the bottom of the gel to remove trapped air bubbles. Electrophoresis can be carded out at 100120 mA/gel (constant amperage) during the day, or as low as 10 mA/gel overnight. Constant voltage or constant power can also be used. Casting and Running Multiple Gradient Gels
Materials for Second Dimension Electrophoresis Multiple casting chambers and electrophoresis chambers for running multiple gels are available from Pierce Apparatus Branch or Hoefer. A power supply capable of reaching 1.5 A is also needed.
Method for Multiple Gel Casting Prepare stock solutions (same as those required for 1D-PAGE gradient gels). Electrode buffer contains 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS. Prepare glass plates by washing in SDS, followed by rinsing with H20, then ethanol. Load the gel-casting chamber with gel plates and seal chamber. Mix together acrylamide and buffers; degas thoroughly. Add 10% SDS stock. Add ammonium persulfate and TEMED immediately before pouring the gradient. A recipe for casting 10 gradient gels designed for the Pace linear gradient maker and the DALT or MEGA
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casting chambers is shown in the tabulation below. (If the size of the casting chamber or the thickness of the gels or glass plates vary, the total volume will have to be adjusted.) Second dimension bis-
Gradient mix
acrylamide stock (ml)
10% 20%
135.0 270.0
10% second
20% second
dimension dimension buffer buffer (ml) (ml) 270.0 0.0
0.0 135.0
10%
10% SDS (ml) 4.0 4.0
ammonium persulfate TEMED (/xl) (~1) 5 2
130 150
Pour 10% gradient mix into the center of the gradient maker and add a stir bar (choose one which almost completely fills diameter of chamber). Turn on the magnetic stirrer until the surface of the acrylamide starts to funnel downward, taking care that air bubbles do not form. Pour 20% gradient mix into the outer well, open the clamps to the casting chamber, and begin pouring the gradient. Immediately open the gradient chambers to allow 20% gradient mix to combine with 10% gradient mix. The acrylamide solution will gently fill the chamber from the bottom. Rotate the DALT or MEGA casting chamber slowly as acrylamide reaches the top corner of the glass plates. When the chamber is almost filled with acrylamide, switch the feed to an incoming line containing glycerol:water (1 : 1) colored with Bromphenol Blue and fill the remainder of the chamber. This glycerol : dye mixture will prevent the acrylamide from polymerizing in the tubing that connects the gradient former to the casting chamber. Spray the surface of the chamber generously with water-saturated
sec-butanol. Allow the gels to polymerize and cool for 1 hr. The gels can then be used immediately or washed and stored at room temperature. For best results, gels should be used soon after casting. (For photographic illustration of this procedure, refer to Ref. 3.)
Loading and Running Multiple Second Dimension Polyacrylamide Gels After polymerization is complete, the gel plates are washed to remove excess acrylamide. Each slab gel is loaded with an IEF gel as described above. (The IEF gels must be sealed on the top of the slab gel with agarose overlay described above.) Electrophoresis chambers for casting and running 10-20 second dimension gels simultaneously are available from Pierce Apparatus Branch,
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Integrated Separation Sciences, Electronucleonics, and Hoefer. Buffer solutions are prepared and added to the tanks. (Note: For convenience, a small volume of buffer may be made from packets of preweighed buffer salts prepared in advance, and the remainder of the volume of water can be added directly to the electrophoresis tanks.) The slab gels are then placed on their sides and are slipped between the rubber spacers. Electrophoresis is carried out for 6-15 hr, depending on the cooling system used, at 70-400 V. Mini-2D-PAGE Samples available in only microgram quantity, or those containing only a limited number of components, may be amenable to mini-2DPAGE analysis. Although the small gel size can severely limit resolution of complex mixtures of proteins, the benefits of mini-2D-PAGE sometimes outweigh the disadvantages. First, the sample size can be reduced to a third of that used on standard-sized 2D-PAGE gels. Second, a complete mini-2D-PAGE analysis, including pouring the gels and staining with Coomassie Blue, can be accomplished in 1 day. Third, both isocratic and gradient gels can be prepared, using the same reagents and power supplies. The specialized equipment needed is not prohibitively expensive and the smaller gels are less costly to pour and stain. Another useful application of minigels (1D or 2D) is to quickly estimate the protein content, purity, and composition of a given sample before running it on a standard 2D-PAGE gel. This step can save valuable time and sample, since it will give dependable information about the volume of sample to be loaded per gel and about the appropriate conditions to yield optimal separation of protein components.
Materials The reagents used for mini-2D-PAGE are the same as those for standard 2D-PAGE. Several companies offer minigel equipment, including Hoefer and Bio-Rad. We have been pleased with the performance of the Bio-Rad modular Mini-Protean II system, and the methods described here will be based on the use of this system. It includes a main buffer chamber which can be used for SDS-PAGE in addition to IEF, electrophoretic transfer and blotting, and electroelution by interchanging modular units. Several gels can be poured at once using the multigel caster, and they can be stored damp and refrigerated in plastic for several days. For casting gradient gels, the small gradient mixers from MRA Corporation (Clearwater, FL) or Bethesda Research Laboratories work well.
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Sample Preparation The same methods for sample preparation are used for mini-2D-PAGE and standard 2D-PAGE. However, the sample solubilization buffer should contain extra Bromphenol Blue to help visualize the sample while loading onto the IEF gel and to ensure that no bubbles are trapped between the gel and the sample. When using SDS solubilization, the volume of solubilization buffer should not exceed 5/zl/IEF gel, or the sample will smear and may crack the gel.
Method for Casting and Running Mini-IEF Gels Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 1.8% bisacrylamide (filter to 0.2/xm) Upper electrode buffer: 0. I N NaOH Lower electrode buffer: 0.06% phosphoric acid The procedure described by the manufacturer can be easily followed, to cast approximately 25 gels at once in capillary tubes, using the following recipe: Urea Ampholytes First dimension bisacrylamide stock H20 Nonidet P-40 10% ammonium persulfate TEMED
4.12 0.50 1.00 3.00 150 35 5
g ml ml ml /xl tzl /xl
Polymerization is complete in 30 min, and the gels should be used within the next 30 min for best results. The directions provided by Bio-Rad can be followed for loading and running mini-IEF gels (up to 16 at a time). The exact amount of sample per gel must be determined experimentally, depending on the composition of your sample. Because of the small gel size and its fragile nature, it is extremely important not to overload the gel. Also, the optimal voltage and time of IEF separation will vary for each sample, and must be determined experimentally. After running, the gels can be easily removed from the tubes, using the syringe adapter from Bio-Rad. They can be ejected directly into a small plastic vial for short-term storage (no longer than 2 weeks, to prevent freezer burn) at - 7 0 °. If second dimension separation is to be done immediately, each IEF gel can be ejected into 0.5 ml of equilibration buffer (as
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in standard 2D-PAGE) and incubated with gentle shaking at room temperature for 10-15 min.
Method for Casting and Running Mini-SDS-PAGE Prepare stock solutions. The stock solutions used are the same as those for standard 2D-PAGE. The Bio-Rad individual gel casting stand can be used for either isocratic or gradient gels, between 0.5 and 1.5 mm thick (1.0-mm-thick gels are required for second dimension separation). Polymerization is complete in only 30 min. Teflon combs are available to form 2-15 wells for SDS-PAGE one-dimensional separation, affording a great deal Of versatility. The following gel recipes are designed to prepare two 1.0-mm SDSPAGE minigels or one 10-20% gradient minigel in the Bio-Rad MiniProtean II casting stand. For separating gels of different height or thickness, volumes must be adjusted. Final acrylamide concentration Component Bisacrylamide stock Running gel buffer H20 TEMED 10% ammonium persulfate
7.5% 3.50 3.48 6.96 6.4 53.0
ml ml ml /zl /zl
10% 4.67 3.48 5.80 6.4 53.0
ml ml ml /LI /.d
10% gradient mix Bisacrylamide stock Running gel buffer H20 TEMED 10% ammonium persulfate
2.33 1.74 2.90 3.2 26.5
ml ml ml tzl /xl
15% 7.00 3.48 3.47 6.4 53.0
20%
ml ml ml gl ~1
9.32 3.48 1.14 6.4 53.0
ml ml ml /zl t~l
20% gradient mix 4.66 1.74 0.57 3.2 26.5
ml ml ml tzl tzl
To load the minigel, the IEF gel is poured out of its tube after equilibration directly onto the side of the minigel glass plate and excess buffer is blotted up. Using a rounded end spatula, the gel can be gently straightened out, parallel to the top of the gel. From one end, the gel is carefully pushed between the glass plates onto the top of the gel, being sure not to trap any bubbles between the gel surfaces. Excess buffer is again blotted away. After assembling the electrophoresis apparatus with the loaded minigels, they are run using conventional power supplies. The Bio-Rad system
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is run at a constant voltage of 200 V. Two minigels will require approximately 60 min of running time. The gels can be stained using Coomassie or silver stain methods, or electrophoretically transferred onto membranes, just like the larger acrylamide gels. Protein Detection Methods in 2D-PAGE In general, the same protein detection methods which are used for one-dimensional PAGE can be used for 2D-PAGE gels (see this volume [33] and [36]; see also Refs. 3-5). We have found, however, that the colorbased silver stain first described by Sammons et al. 7 and outlined in detail by Dunbar 3 is easiest to use and gives optimal protein resolution in 2DPAGE. This stain results in vivid colors (unlike other silver stains which give less color) that are extremely important in identifying proteins and protein families and in comparing protein patterns. It is frequently desirable to first stain gels with the Coomassie Blue method to visualize the most abundant proteins, and then restain the same gel with the colorbased silver stain to visualize the less abundant proteins (after thoroughly destaining). Quantitation of Proteins in 2D-PAGE Advances in the methods for 2D-PAGE separation of proteins have been accompanied by the development of computer systems to analyze the resulting protein patterns, and to quantitate the individual protein components. 8-H Hardware and software are available commercially from a wide variety of sources. These data analysis systems range from simple inexpensive programs for personal computers to more expensive systems which allow the simultaneous analysis and comparison of complex protein patterns in 2D-PAGE gels. The accuracy and precision of the information generated by a computer analysis package depend on both the type of scanning hardware and the quality of the data manipulation soft7 D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 3, 135 (1981). s j. I. Garrels, J. T. Farrar, and C. B. Burwell, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 38. Academic Press, New York, 1984. 9 L. E. Lipkin and P. F. Lemkin, Clin. Chem. 26, 1403 (1980). 10 D. W. Sammons, L. D. Adams, T. J. Vidmar, C. A. Hatfield, D. H. Jones, P. J. Chubb, and S. W. Crooks, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 112. Academic Press, New York, 1984. H j. Taylor, N. L. Anderson, and N. G. Anderson, Electrophoresis 3, 338 (1983).
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ware. Therefore, it is important to determine what qualitative and quantitative information is needed to interpret the data, 8 and to choose the hardware and software most appropriate for that application. If a large number of 2D-PAGE gels will be processed on a routine basis, a sophisticated shared instrumentation computer system which can analyze gels rapidly and accurately and which is "user friendly" is advisable. We have used one such system, the BioImage Visage computer analysis system, and have been pleased with its performance. Before subjecting 2D-PAGE gels to computer analysis there are several points to consider. First, computer quantitation of poor quality gels is of limited value. Therefore, the 2D-PAGE separation of components in a complex protein mixture must be optimized prior to analysis. Second, the information obtained from computer "quantitation" is relative to the method of protein detection used, and thus to the nature of the proteins themselves. For example, the autoradiographic signal generated by [35S]methionine-labeled proteins will be proportional to the number of methionine residues in the protein and not necessarily to the amount of that protein present in the sample. Finally, useful information can be gathered from visual inspection of reproducible, high-quality 2D-PAGE gels without the assistance of computer programs. The lack of a computer system for analysis should not be a major factor in considering the use of 2D-PAGE. Troubleshooting in 2D-PAGE Because of the complex nature of 2D-PAGE methods, there are technical problems that are frequently encountered while conditions are being optimized for a particular sample. 3,~2,~3A summary of these problems and some suggestions for resolving them are presented in Table I. If the described protocols are followed precisely, only high-quality reagents are used, and care is taken to properly prepare the sample, the 2D-PAGE protein separation and resolution should be excellent. Strategies to Optimize 2D-PAGE Resolution The 2D-PAGE methods described in this chapter are standard procedures widely used by different laboratories that enable the direct compari12 J. VanBlerkom, in "Methods in Mammalian Reproduction" (J. C. Daniel, Jr., ed.), p. 67. Academic Press, New York, 1978. 13 R. Bravo, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 4. Academic Press, New York, 1984.
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TABLE I COMMON PROBLEMSASSOCIATEDWITH 2D-PAGE Problem First dimension Gels do not polymerize
Gels break during focusing
Possible causes
Solutions
Inferior, aged, or improperly prepared catalysts; impure reagents containing contaminants which interfere with polymerization Hole in pH gradient
Start over with fresh, highquality reagents, and check pipetting measurements
Gel is overloaded Gels fall out of tube during focusing Gels will not come out of tubes after focusing Poor separation of proteins
Too much NP-40 in gel
Tubes are not cleaned properly
Improper ampholyte pH range Insufficient protein solubilization Nucleic acid contamination
IEF patterns vary from day to day
Second dimension Irregular gradients
Changing electrofocusing time
Mix ampholytes from two different sources Reduce amount of protein loaded on gel Measure using clipped off pipet tip and wipe outside surface of tip Wash tubes with Chromerge and rinse with water only; do not use methanol or siliconizing solutions Try another pH range Increase ratio of solubilization reagent to protein sample If gels are to be silver stained, increase amount of solubilization reagent and ultracentrifuge at 2 x 105 g. If proteins are to be detected by autoradiography, add nuclease preparations to sample Standardize voltage-hours
Different lots or sources of ampholytes used Stock reagents are too old Inadequate solubilization
Standardize ampholyte source Prepare fresh reagents Increase amount of solubilization reagent
Improper equipment or technique; acrylamide polymerization is too fast
Decrease slightly the amount of catalyst
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TWO-DIMENSIONAL GEL ELECTROPHORESIS TABLE I (continued) Problem
Possible causes
"Fuzzy" protein spots
IEF gel was equilibrated too long before loading onto second dimension gel; not optimal acrylamide concentration in SDS gel Nucleic acid contamination of protein sample Improper or incomplete solubilization
Streaking
Gel is overloaded Sample may contain glycoproteins, which often streak in PAGE
Solutions Use a gradient gel, or vary the acrylamide concentration slightly See First Dimension, poor separation of proteins Increase amount of solubilization reagent, or change type of reagent used Reduce amount of protein on gel Reduce amount of sample on gel, or deglycosylate glycoproteins
son of protein patterns independently generated by individual research groups. It is occasionally necessary, however, to modify these procedures for unusual proteins or for a protein which is extensively posttranslationally modified. F o r example, mixing ampholytes of different ranges may improve resolution of proteins within a particular pH range. Also, protein samples containing an abundance of one protein can alter the p H range of the ampholytes themselves. Therefore, it may be necessary to c o m p e n s a t e by adding different ranges of ampholytes to the isoelectric focusing dimension. When carrying out the initial 2D-PAGE analysis of any protein mixture, it is helpful to include a lane for one-dimensional S D S - P A G E separation of the original sample on the same second dimension polyacrylamide gel. This will determine if any of the protein components are outside the range of the ampholytes used for IEF. Depending on the sample and the complexity of the protein pattern, it may be necessary to try different solubilization conditions, different ampholyte ranges, or N E P H G E (nonequilibrium p H gradient electrophoresis) gels to establish the best method to resolve all the proteins of interest. Standardization of 2 D - P A G E The standardization of 2D-PAGE methods has b e c o m e more important as the need for interlaboratory comparisons of protein patterns has
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IEF 9
4M r x 10 -3
m
t/) 0 =
O I'll
FIG. 1. Example of cellular proteins resolved by high-resolution two-dimensional polyacrylamide gel electrophoresis using color-based silver stain 3,7 illustrating the standardized method for presenting 2D-PAGE protein patterns.
increased. As discussed above, the sophisticated equipment now available for running and analyzing two-dimensional polyacrylamide gels has advanced the standardization process. Another factor enabling accurate comparisons among gels is the use of internal standards for both charge and molecular weight separations. (The procedures which measure pH directly in the gels or in gel slices have proved to be totally inadequate and not reproducible.) Proteins which are modified by carbamylation have proved to be excellent charge standardsy and numerous types of molecular weight standards are commercially available. Publication Format of 2D-PAGE Patterns The protein patterns obtained by 2D-PAGE can be extremely complicated. However, reproducible protein patterns can easily be recognized if they are presented in a standardized format. Many different laboratories, in conjunction with the International Electrophoresis Society, have agreed on a useful presentation format: the acidic end of the IEF gel at the left, and the basic end at the right; i.e., low pH values at the left, increasing toward the right. The second dimension separation is oriented with low-molecular-weight proteins at the bottom and high molecular weights
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at the top, which is standard for 1D-PAGE analysis. The presentation of such a gel is illustrated in Fig. I. This format is now required by the Journal of Electrophoresis and will likely be required by other journals in the future. Acknowledgments The authors wish to acknowledgethe numeroustechniciansand graduate students who have assisted in the developmentof these techniquesover the years. We thank Drs. N. L. Anderson, S. Tollaksen, and D. Sammonsfor manyfruitfuldiscussions,and Ms. Suzanne Mascola for expert secretarial assistance.
[35] I s o e l e c t r i c F o c u s i n g
By DAVID E. GARFIN Proteins, as amphoteric molecules, carry positive, negative, or zero net charges depending on the pH of their local environments. The overall charge of a particular protein is determined by the ionizable acidic and basic side chains of its constituent amino acids and prosthetic groups. Carboxylic acid groups (--COOH) in proteins are uncharged in acidic solutions and dissociate to the anionic form ( - - C O 0 - ) at higher pH values, above about pH 3. Amines (--NH2) and other basic functions of proteins, such as guanidines, are uncharged at alkaline pH, but are cationic below about pH 10 (e.g., --NH3+). The pH at which individual ionizable side chains actually dissociate is affected by the overall composition of the protein and the properties of the medium. As a result, each individual ionizable group in a protein has a nearly unique dissociation point. The net charge on a protein is the algebraic sum of all its positive and negative charges. There is, thus, a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed pl, is a characteristic physicochemical property of every protein. If the number of acidic groups in a protein exceeds the number of basic groups, the pl of that protein will be at a low pH value. If, on the other hand, basic groups outnumber acidic groups, the pI will be high. Proteins show considerable variation in isoelectric points, but pl values usually fall in the range of p H 3 t o p H 10. Proteins are positively charged in solutions at pH values below their pl values and negatively charged above their isoelectric points. In electroMETHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Copper staining31 (above) is advisable for the visualization of the bands in preparative SDS-PAGE, since this method does not employ fixative solvents. Desired bands are cut from the gel and destained by incubation in three changes (for 10 min each) of 0.25 M EDTA, 0.25 M Tris-Cl, pH 9. After destaining, gel slices are incubated in the appropriate elution buffer. Proteins are often extracted from macerated gel slices by simple diffusion into appropriate buffers or by solubilization of the gel. 5,33In the latter method, cross-linkers other than bisacrylamide are copolymerized into the gels. 5,7 For example, gels cross-linked with N,N'-bisacrylylcystamine (BAC) are dissolvable in 2-mercaptoethanol or dithiothreitol, while both N,N'-dihydroxyethylenebisacrylamide (DHEBA) and N,N'-diallyltartardiamide (DATD) result in gels which can be solubilized with periodic acid. Once gels have been dissolved, proteins must be separated from the large excess of gel material by gel filtration or ion-exchange chromatography. Electrophoretic elution is an efficient method for recovering proteins from gel slices. 2,5,8 In the simplest versions of this method, proteins are electrophoresed out of gel pieces into dialysis sacks in the types of apparatus used for running cylindrical gel rods. Devices are available for the rapid recovery of proteins in small volumes with yields of greater than 70% in most cases. Elution takes about 3 hr at 10 mA/tube in 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3 (standard SDS-PAGE electrode buffer). SDS can be removed from the eluted samples by dialysis or ionexchange chromatography)4 34A. J. Furth, Anal. Biochem. 109, 207 (1980).
[34] P r o t e i n Analysis U s i n g H i g h - R e s o l u t i o n T w o - D i m e n s i o n a l P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s By BONNIE S. DUNBAR, HITOMI KIMURA, and THERESE M. TIMMONS
The term two-dimensional electrophoresis has been used to describe a variety of methods employing separation of molecules in two dimensions. The term high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now specifically applied to the separation of proteins in the first dimension according to their isoelectric points using isoelectric focusing (IEF) with carrier ampholytes after reduction of disulfide bonds, METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
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followed by separation in the second dimension according to their molecular weights using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as defined by O'Farrell. 1 The history of the major developments in the field of electrophoresis and 2D-PAGE has been summarized in detail elsewhere. 2-4 The most significant recent advances in this technology have come through the standardization of equipment and reagents and the simplification of equipment for reproducible analysis and large scale analyses. 3,5 Because large numbers of laboratories are now using these standardized procedures, they are the methods described in this chapter. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. The use of 2D-PAGE has become increasingly popular during the past decade. Two-dimensional PAGE allows the resolution of a complex protein mixture into more discrete components than 1D-PAGE since it separates on the basis of protein charge in addition to molecular weight. The major advantage of large-scale 2D-PAGE is the improvement in reproducibility of protein patterns. This enables the researcher to directly compare the analyses of complex protein mixtures, whether the 2D-PAGE separations are conducted simultaneously or in different experiments. This feature makes 2D-PAGE a versatile and powerful tool in both basic and clinical research. Applications of 2D-PAGE The most common uses of 2D-PAGE are the analysis of complex mixtures of proteins and the analysis of the posttranslational modification of proteins. 2D-PAGE can also provide valuable information about the molecular properties of proteins, including an estimate of the relative isoelectric points (pI) and molecular weights 3 of proteins. However, it is generally inadequate to use this as the sole method for the precise determination of these parameters. For example, the disulfide bonds of the i p. H. O'Farrell and J. I. Garrels, this series, Vol. 100, p. 411. 2 N. G. Anderson and L. Anderson, Clin. Chem. 28, 739 (1982). 3 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. 4 B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C., 1981. L. Anderson, "Two-Dimensional Electrophoresis: Operation of the ISO-DALT System." Large Scale Biology Press, Washington, D.C., 1988.
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proteins analyzed by 2D-PAGE are usually reduced so the protein patterns may reflect subunit peptides. The pI and molecular weight values observed may therefore be different from those of the native proteins. One should be careful not to overinterpret data obtained from electrofocusing and 2D-PAGE. Another common use of 2D-PAGE is to rapidly purify a specific protein which can be cut from the gel and used directly to obtain amino acid sequence or to purify antibodies. These antibodies can then be used to immunoaffinity purify the original protein in quantities sufficient for detailed chemical characterization. Immunoblotting using antibodies to detect antigens separated by 2DPAGE also provides an excellent method to analyze antibody specificity and to analyze carbohydrate or other epitopes. Finally, the use of 2D-PAGE with silver staining provides one of the best methods to estimate protein purity. This analysis, in conjunction with one-dimensional analysis of proteins visualized by silver stain (to detect proteins whose pI is outside the pH range of the ampholines), will provide a rigorous estimate of protein purity. Sample Preparation and Solubilization Procedures The preparation of samples for 2D-PAGE analysis is the most critical step in guaranteeing excellent reproducible results. All tissues and samples should be handled in the cold and stored at -70 °. It is important that the ratio of solubilization buffer to protein concentration be optimized for each sample. We have found the following ratios to be adequate for most samples: (1) 200-500/zg tissue homogenate/2 ml solubilization buffer, (2) 20-50/xl cell pellet/300/zl solubilization buffer, (3) 1 x 10 6 cells in tissue culture plate with 500/zl solubilization buffer to solubilize cells directly, and (4) 10-200/xg soluble protein/30-50/zl solubilization buffer. Note: 550/zl of each of the above samples should be adequate for identification of abundant proteins by Coomassie Blue staining or of minor proteins by silver staining in two-dimensional gels. Materials
Sodium dodecyl sulfate (SDS) (Bio-Rad, Richmond, CA) Cyclohexylaminoethane (CHES) (Calbiochem, San Diego, CA) Glycerol (Fisher, Pittsburgh, PA) 2-Mercaptoethanol (Bio-Rad) Urea (ultrapure) (Bio-Rad) Nonidet P-40 (nonionic detergent, NP-40) (Accurate Chemical, Westbury, NY)
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Ampholytes [pH 3.5-10: Bio-Rad, LKB, or Pharmacia (Piscataway, NJ): pH 2-11: Serva, Garden City Park, NY]. This wide-range mixture is appropriate for most routine samples. Other pH range or combinations of brands of ampholytes may be used in some instances H20, deionized with mixed bed resin (Continental filter system) or deionized double-distilled H20 Method
The two solubilization buffers which can be used for isoelectric focusing are as follow: SDS solubilization solution: 0.05 M CHES, 2% SDS, 10% glycerol, small amount of Bromphenol Blue, pH 9.5. Add 2% 2-mercaptoethanol just before use. Samples should be suspended in SDS solubilization buffer, placed in a tightly capped glass vial, and heated for 510 min in a boiling water bath. (Thick plastic tubes such as microfuge tubes are insulated and interfere with heating.) It may be necessary to solubilize some samples at room temperature for 2-3 hr, with or without heating Urea solubilization solution: 9 M urea, 4% Nonidet P-40. Add 2% 2mercaptoethanol and 2% ampholytes to a small aliquot of solubilization buffer just prior to use. These reagents should be filtered to 0.2 /zm with a syringe filter for best results. Samples should be suspended in the urea solubilization solution and incubated at room temperature for 2 hr. Caution: Do not heat, or you will generate charge artifacts Following the incubation, samples are centrifuged to remove nonsolubilized material and nucleic acids that may interfere with focusing or cause streaking in second-dimension protein patterns (100,000-200,000 g for 2 hr is suggested). We recommend using a Beckman Ti-42.2 rotor, which holds 72 tubes. Isoelectric Focusing Materials
Urea (ultrapure) (Bio-Rad) Ampholytes (LKB, Serva, or Pharmacia recommended); pH will depend on needs of investigator Acrylamide (Bio-Rad) Bisacrylamide (Bio-Rad) Nonidet P-40 (Accurate) Ammonium persulfate (Bio-Rad)
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N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad) Sodium hydroxide (Sigma, St. Louis, MO) Phosphoric acid (Fisher) Chromerge (Fisher) Glass pipet tubes (0.2 ml disposable) (American Scientific Products, S/P disposable serological pipets, 0.2 ml, #P4644-2T) Gel electrophoresis apparatus: Any tube gel electrophoresis apparatus can be used if appropriate grommets or corks are prepared to fit small tubes (e.g., Bio-Rad electrophoresis unit model 175 tube gel apparatus). Alternatively, multiple IEF casting systems now available from Pierce Apparatus Branch, Hoefer Scientific, and Integrated Separation Sciences have been optimized for these procedures and are highly recommended Method To cast IEF gels, add urea (8.25 g) to 6 ml HzO plus 2.0 ml acrylamide stock (30 g acrylamide : 1.8 g bisacrylamide : 100 ml HzO, filtered to 0.2 tzm). Dissolve the urea in the H20 by swirling the flask under warm running water. Caution: Do not heat solution. Add ampholytes (0.75 ml) to the mixture of acrylamide, water, and urea, swirl the solution gently to mix, and degas on a lyophilizer. Add 0.3 ml Nonidet P-40, and mix gently. (Hint: A large, plastic Eppendorf pipet tip can be cut off for easier and more accurate pipetting of viscous detergents.) Add ammonium persulfate (70 txl of a 10% solution) and TEMED (10/zl) to the acrylamide solution, and swirl the flask gently to mix. Cast IEF gels to a height of approximately 12 cm using capillary action, by overlayering acrylamide stock with water using a commercial casting apparatus, or a home-made casting chamber prepared from 2- to 50-ml plastic conical centrifuge tubes (as described in Dunbar3). Allow 1 hr for polymerization, and place tubes into the electrophoresis chamber. Prepare upper electrode buffer (0.02 N NaOH degassed thoroughly on lyophilizer) and lower electrode buffer (0.085% phosphoric acid), and add to chamber. Prefocus the gels at 200 V for 1-2 hr. In theory, this will remove ions which may interfere with the focusing. We have frequently omitted this step, however, with no noticeable differences in protein patterns. Load the protein samples (5-50 ~1) with a Hamilton syringe under the upper electrode buffer. Carry out isoelectric focusing for 10,000-12,000 V-hr (e.g., 17 hr at 700 V). The optimal conditions will depend on the nature of your sample and the dimensions and volume of your IEF gels. We have found that resolution of proteins is sharper if you focus for a shorter period of time at higher voltage (i.e., 700 V for 16 hr is better than 500 V for 22 hr).
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To remove gel, insert a yellow Eppendorf pipet tip attached to a 3-ml syringe filled with water into the top of the tube, and gently push out the gel. The IEF gels are equilibrated in buffer (0.125 M Tris-base; 2% SDS; 10% glycerol, pH 6.8, plus 0.2-0.8% 2-mercaptoethanol added just before use) for 15 min, to remove ampholytes and urea and to recoat the proteins with SDS. In some instances, we have equilibrated the gels for as little as 2-5 min with excellent results. Note: The "mercaptan" artifact commonly observed by silver staining which appears as two distinct lines having molecular weights of approximately 50K and 70K can be reduced if little or no 2-mercaptoethanol is used in the equilibration buffer. You should first establish whether the omission of this reducing agent will alter your protein patterns by comparing samples run in its presence or absence. The IEF gel can be frozen at - 7 0 ° before equilibration, and thawed in equilibration buffer immediately before placing on the surface of the second dimension slab gel. Nonequilibrium pH Gradient Electrophoresis (NEPHGE Gel System) in 2D-PAGE "Nonequilibrium" isoelectric focusing techniques are especially useful for the first dimension separation of basic proteins, which are not well resolved or cannot be resolved by other IEF procedures. 6 Samples must be solubilized in the urea solubilization buffer above. All gel-casting procedures should be carried out as for standard equilibrium IEF, except that the upper and lower buffers are reversed: the upper electrode buffer should contain phosphoric acid, and the lower buffer should contain sodium hydroxide. When attaching the electrodes to the power supply, be sure to attach the upper buffer reservoir to the positive electrode and the lower buffer reservoir to the negative electrode. Finally, the IEF gels should be removed at intervals such as 2000, 4000, 6000, or 8000 V-hr. Total volt-hours will have to be optimized to resolve different proteins of interest, since this is a nonequilibrium system. Casting and Running Individual One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gels for Second Dimension Electrophoresis Materials
Acrylamide (Bio-Rad, Polysciences, Serva Fine Chemicals, or Sigma): Reagents from the latter two sources are less expensive, but require filtering through Whatman #3 filter paper, followed by a 0.2-~ Milli6 p. Z. O'Farrell, H. M. G o o d m a n , a n d P. H. O'Farrell, Cell 12, 1133 (1977).
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pore filter. These impure reagents may also have contaminants detectable by silver stain. They can easily be distinguished from protein spots in 2D-PAGE, but will make analysis of silver-stained 1D-PAGE difficult N,N'-Methylene(bis)acrylamide (Bio-Rad) Trizma base (Sigma) Glycine (Sigma) SDS (Bio-Rad) 2-Mercaptoethanol (Bio-Rad) Glycerol (Fisher) N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad) sec-Butanol (Fisher) Agarose (Bio-Rad) Glass plates and spacers for individual or multiple gel-casting systems: The size plates will depend on the type of electrophoresis chamber that will be utilized (e.g., 18 x 16 cm plates with 1.5-cm spacers are compatible with Bio-Rad or Hoefer electrophoresis units). The recipes in this chapter are for this size gel Electrophoresis chambers: These can be obtained commercially from Bethesda Research Laboratories, Bio-Rad, Hoefer, or can be custom made (Studier apparatus) Gradient maker (double conical style recommended) Multiple electrophoresis gradient gel-casting systems (highly recommended if you do gels regularly; greatly improves reproducibility!)
Method for Casting Nongradient Gels Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide (filter to 0.2 t~m) Gel buffer stock: 1.5 M Trizma base, 0.4% SDS (filter to 0.2/~m) Ammonium Persulfate: 10 g ammonium persulfate; final volume 100 ml (filter to 0.2 t~m). Freeze at - 2 0 ° in small aliquots to guarantee the consistency of polymerization for as long as the stock lasts Tank buffer: 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS Assemble the gel-casting apparatus. Combine acrylamide, buffer, and H20; degas (see tabulation below for final acrylamide concentration). Add TEMED and mix thoroughly but gently by swirling the beaker. Add ammonium persulfate and swirl gently to mix. Pour the mixture down one edge of the spacer of the gel-casting unit using a 25-ml pipet, or a syringe and a large (18-gauge) needle. Fill to within 3 cm of the top of the glass plates. Carefully overlay with water-saturated sec-butanol, and allow to
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polymerize for 45-60 min. (Note: This is the most important step for obtaining good resolution in slab polyacrylamide gels.) When the gel is polymerized, rinse the surface several times with distilled water, and drain well. Final acrylamide concentration Component
7.5%
10%
12.5%
15%
Bisacrylamide stock Gel buffer stock H20 10% ammonium persulfate TEMED
7.1 ml 7.1 ml 14.2 ml 105/.tl 15/zl
9.5 ml 7.1 ml 11.8 ml 105/~1 15/zl
11.8 ml 7. I ml 9.5 ml 105/~1 15/~1
14.2 ml 7.1 ml 7.1 ml 105/~1 15/zl
Method for Casting Gradient Gels If silver staining methods are to be used, all reagents should be filtered to 0.2/zm. Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide Second dimension buffer stock: 40 g Trizma base, 20 g Trizma-HCl; final volume 300 ml, pH 8.5-8.6 10% second dimension buffer: Three parts second dimension buffer stock plus five parts H 2 0 20% second dimension buffer: Three parts second dimension buffer stock plus one part glycerol 10% SDS: 10 g SDS; final volume 100 ml Assemble individual slab gel units in casting apparatus. For each gradient gel (approximately 40 ml/volume), prepare the reagents as per the following tabulation:
Gradient mix
Second dimension bisacrylamide stock (ml)
10% second dimension buffer (ml)
20% second dimension buffer (ml)
10% SDS (ml)
10% ammonium persulfate (gl)
10% 20%
6.3 14.7
14.7 0.0
0.0 6.3
0.2 0.2
30 15
TEMED (/xl) 2
2
Place 20% gradient mix in the internal .chamber of the gradient maker and begin mixing with a magnetic stir bar. Place 10% gradient mix in the
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external chamber, start the peristaltic pump, and open the gradient chamber to begin gradient formation. When the gradient is finished, spray the surface of the gel with water-saturated sec-butanol. After polymerization is complete, rinse the surface of the gel with HE0 and drain well.
Loading and Running Individual Second Dimension Polyacrylamide Gels Set the polymerized slab gel on a loading stand and lay the IEF gel on a platform (or sheet of parafilm) and gently straighten out. Allow the gel to slide into place along the surface, being sure not to trap air bubbles. If desired, seal the IEF gel with a small amount of overlay agarose (0.25 M Trizma base, 0.192 M glycine, 0.1% SDS, and 0.5% agarose heated to dissolve thoroughly, and then cooled slightly before overlayering gel). If standard electrophoresis chambers are used (e.g., Studier, Bio-Rad, or Hoefer electrophoresis apparatus), electrophoresis is carried out by placing the electrode buffer in the upper and lower chambers. The slab gels are then placed into these chambers, taking care to avoid air bubbles being trapped at the bottom of the slab acrylamide gel. This can be done by tilting the gel as it is lowered into the chamber and by tilting the chamber so that the buffer will move across the bottom of the gel to remove trapped air bubbles. Electrophoresis can be carded out at 100120 mA/gel (constant amperage) during the day, or as low as 10 mA/gel overnight. Constant voltage or constant power can also be used. Casting and Running Multiple Gradient Gels
Materials for Second Dimension Electrophoresis Multiple casting chambers and electrophoresis chambers for running multiple gels are available from Pierce Apparatus Branch or Hoefer. A power supply capable of reaching 1.5 A is also needed.
Method for Multiple Gel Casting Prepare stock solutions (same as those required for 1D-PAGE gradient gels). Electrode buffer contains 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS. Prepare glass plates by washing in SDS, followed by rinsing with H20, then ethanol. Load the gel-casting chamber with gel plates and seal chamber. Mix together acrylamide and buffers; degas thoroughly. Add 10% SDS stock. Add ammonium persulfate and TEMED immediately before pouring the gradient. A recipe for casting 10 gradient gels designed for the Pace linear gradient maker and the DALT or MEGA
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casting chambers is shown in the tabulation below. (If the size of the casting chamber or the thickness of the gels or glass plates vary, the total volume will have to be adjusted.) Second dimension bis-
Gradient mix
acrylamide stock (ml)
10% 20%
135.0 270.0
10% second
20% second
dimension dimension buffer buffer (ml) (ml) 270.0 0.0
0.0 135.0
10%
10% SDS (ml) 4.0 4.0
ammonium persulfate TEMED (/xl) (~1) 5 2
130 150
Pour 10% gradient mix into the center of the gradient maker and add a stir bar (choose one which almost completely fills diameter of chamber). Turn on the magnetic stirrer until the surface of the acrylamide starts to funnel downward, taking care that air bubbles do not form. Pour 20% gradient mix into the outer well, open the clamps to the casting chamber, and begin pouring the gradient. Immediately open the gradient chambers to allow 20% gradient mix to combine with 10% gradient mix. The acrylamide solution will gently fill the chamber from the bottom. Rotate the DALT or MEGA casting chamber slowly as acrylamide reaches the top corner of the glass plates. When the chamber is almost filled with acrylamide, switch the feed to an incoming line containing glycerol:water (1 : 1) colored with Bromphenol Blue and fill the remainder of the chamber. This glycerol : dye mixture will prevent the acrylamide from polymerizing in the tubing that connects the gradient former to the casting chamber. Spray the surface of the chamber generously with water-saturated
sec-butanol. Allow the gels to polymerize and cool for 1 hr. The gels can then be used immediately or washed and stored at room temperature. For best results, gels should be used soon after casting. (For photographic illustration of this procedure, refer to Ref. 3.)
Loading and Running Multiple Second Dimension Polyacrylamide Gels After polymerization is complete, the gel plates are washed to remove excess acrylamide. Each slab gel is loaded with an IEF gel as described above. (The IEF gels must be sealed on the top of the slab gel with agarose overlay described above.) Electrophoresis chambers for casting and running 10-20 second dimension gels simultaneously are available from Pierce Apparatus Branch,
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Integrated Separation Sciences, Electronucleonics, and Hoefer. Buffer solutions are prepared and added to the tanks. (Note: For convenience, a small volume of buffer may be made from packets of preweighed buffer salts prepared in advance, and the remainder of the volume of water can be added directly to the electrophoresis tanks.) The slab gels are then placed on their sides and are slipped between the rubber spacers. Electrophoresis is carried out for 6-15 hr, depending on the cooling system used, at 70-400 V. Mini-2D-PAGE Samples available in only microgram quantity, or those containing only a limited number of components, may be amenable to mini-2DPAGE analysis. Although the small gel size can severely limit resolution of complex mixtures of proteins, the benefits of mini-2D-PAGE sometimes outweigh the disadvantages. First, the sample size can be reduced to a third of that used on standard-sized 2D-PAGE gels. Second, a complete mini-2D-PAGE analysis, including pouring the gels and staining with Coomassie Blue, can be accomplished in 1 day. Third, both isocratic and gradient gels can be prepared, using the same reagents and power supplies. The specialized equipment needed is not prohibitively expensive and the smaller gels are less costly to pour and stain. Another useful application of minigels (1D or 2D) is to quickly estimate the protein content, purity, and composition of a given sample before running it on a standard 2D-PAGE gel. This step can save valuable time and sample, since it will give dependable information about the volume of sample to be loaded per gel and about the appropriate conditions to yield optimal separation of protein components.
Materials The reagents used for mini-2D-PAGE are the same as those for standard 2D-PAGE. Several companies offer minigel equipment, including Hoefer and Bio-Rad. We have been pleased with the performance of the Bio-Rad modular Mini-Protean II system, and the methods described here will be based on the use of this system. It includes a main buffer chamber which can be used for SDS-PAGE in addition to IEF, electrophoretic transfer and blotting, and electroelution by interchanging modular units. Several gels can be poured at once using the multigel caster, and they can be stored damp and refrigerated in plastic for several days. For casting gradient gels, the small gradient mixers from MRA Corporation (Clearwater, FL) or Bethesda Research Laboratories work well.
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Sample Preparation The same methods for sample preparation are used for mini-2D-PAGE and standard 2D-PAGE. However, the sample solubilization buffer should contain extra Bromphenol Blue to help visualize the sample while loading onto the IEF gel and to ensure that no bubbles are trapped between the gel and the sample. When using SDS solubilization, the volume of solubilization buffer should not exceed 5/zl/IEF gel, or the sample will smear and may crack the gel.
Method for Casting and Running Mini-IEF Gels Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 1.8% bisacrylamide (filter to 0.2/xm) Upper electrode buffer: 0. I N NaOH Lower electrode buffer: 0.06% phosphoric acid The procedure described by the manufacturer can be easily followed, to cast approximately 25 gels at once in capillary tubes, using the following recipe: Urea Ampholytes First dimension bisacrylamide stock H20 Nonidet P-40 10% ammonium persulfate TEMED
4.12 0.50 1.00 3.00 150 35 5
g ml ml ml /xl tzl /xl
Polymerization is complete in 30 min, and the gels should be used within the next 30 min for best results. The directions provided by Bio-Rad can be followed for loading and running mini-IEF gels (up to 16 at a time). The exact amount of sample per gel must be determined experimentally, depending on the composition of your sample. Because of the small gel size and its fragile nature, it is extremely important not to overload the gel. Also, the optimal voltage and time of IEF separation will vary for each sample, and must be determined experimentally. After running, the gels can be easily removed from the tubes, using the syringe adapter from Bio-Rad. They can be ejected directly into a small plastic vial for short-term storage (no longer than 2 weeks, to prevent freezer burn) at - 7 0 °. If second dimension separation is to be done immediately, each IEF gel can be ejected into 0.5 ml of equilibration buffer (as
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in standard 2D-PAGE) and incubated with gentle shaking at room temperature for 10-15 min.
Method for Casting and Running Mini-SDS-PAGE Prepare stock solutions. The stock solutions used are the same as those for standard 2D-PAGE. The Bio-Rad individual gel casting stand can be used for either isocratic or gradient gels, between 0.5 and 1.5 mm thick (1.0-mm-thick gels are required for second dimension separation). Polymerization is complete in only 30 min. Teflon combs are available to form 2-15 wells for SDS-PAGE one-dimensional separation, affording a great deal Of versatility. The following gel recipes are designed to prepare two 1.0-mm SDSPAGE minigels or one 10-20% gradient minigel in the Bio-Rad MiniProtean II casting stand. For separating gels of different height or thickness, volumes must be adjusted. Final acrylamide concentration Component Bisacrylamide stock Running gel buffer H20 TEMED 10% ammonium persulfate
7.5% 3.50 3.48 6.96 6.4 53.0
ml ml ml /zl /zl
10% 4.67 3.48 5.80 6.4 53.0
ml ml ml /LI /.d
10% gradient mix Bisacrylamide stock Running gel buffer H20 TEMED 10% ammonium persulfate
2.33 1.74 2.90 3.2 26.5
ml ml ml tzl /xl
15% 7.00 3.48 3.47 6.4 53.0
20%
ml ml ml gl ~1
9.32 3.48 1.14 6.4 53.0
ml ml ml /zl t~l
20% gradient mix 4.66 1.74 0.57 3.2 26.5
ml ml ml tzl tzl
To load the minigel, the IEF gel is poured out of its tube after equilibration directly onto the side of the minigel glass plate and excess buffer is blotted up. Using a rounded end spatula, the gel can be gently straightened out, parallel to the top of the gel. From one end, the gel is carefully pushed between the glass plates onto the top of the gel, being sure not to trap any bubbles between the gel surfaces. Excess buffer is again blotted away. After assembling the electrophoresis apparatus with the loaded minigels, they are run using conventional power supplies. The Bio-Rad system
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is run at a constant voltage of 200 V. Two minigels will require approximately 60 min of running time. The gels can be stained using Coomassie or silver stain methods, or electrophoretically transferred onto membranes, just like the larger acrylamide gels. Protein Detection Methods in 2D-PAGE In general, the same protein detection methods which are used for one-dimensional PAGE can be used for 2D-PAGE gels (see this volume [33] and [36]; see also Refs. 3-5). We have found, however, that the colorbased silver stain first described by Sammons et al. 7 and outlined in detail by Dunbar 3 is easiest to use and gives optimal protein resolution in 2DPAGE. This stain results in vivid colors (unlike other silver stains which give less color) that are extremely important in identifying proteins and protein families and in comparing protein patterns. It is frequently desirable to first stain gels with the Coomassie Blue method to visualize the most abundant proteins, and then restain the same gel with the colorbased silver stain to visualize the less abundant proteins (after thoroughly destaining). Quantitation of Proteins in 2D-PAGE Advances in the methods for 2D-PAGE separation of proteins have been accompanied by the development of computer systems to analyze the resulting protein patterns, and to quantitate the individual protein components. 8-H Hardware and software are available commercially from a wide variety of sources. These data analysis systems range from simple inexpensive programs for personal computers to more expensive systems which allow the simultaneous analysis and comparison of complex protein patterns in 2D-PAGE gels. The accuracy and precision of the information generated by a computer analysis package depend on both the type of scanning hardware and the quality of the data manipulation soft7 D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 3, 135 (1981). s j. I. Garrels, J. T. Farrar, and C. B. Burwell, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 38. Academic Press, New York, 1984. 9 L. E. Lipkin and P. F. Lemkin, Clin. Chem. 26, 1403 (1980). 10 D. W. Sammons, L. D. Adams, T. J. Vidmar, C. A. Hatfield, D. H. Jones, P. J. Chubb, and S. W. Crooks, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 112. Academic Press, New York, 1984. H j. Taylor, N. L. Anderson, and N. G. Anderson, Electrophoresis 3, 338 (1983).
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ware. Therefore, it is important to determine what qualitative and quantitative information is needed to interpret the data, 8 and to choose the hardware and software most appropriate for that application. If a large number of 2D-PAGE gels will be processed on a routine basis, a sophisticated shared instrumentation computer system which can analyze gels rapidly and accurately and which is "user friendly" is advisable. We have used one such system, the BioImage Visage computer analysis system, and have been pleased with its performance. Before subjecting 2D-PAGE gels to computer analysis there are several points to consider. First, computer quantitation of poor quality gels is of limited value. Therefore, the 2D-PAGE separation of components in a complex protein mixture must be optimized prior to analysis. Second, the information obtained from computer "quantitation" is relative to the method of protein detection used, and thus to the nature of the proteins themselves. For example, the autoradiographic signal generated by [35S]methionine-labeled proteins will be proportional to the number of methionine residues in the protein and not necessarily to the amount of that protein present in the sample. Finally, useful information can be gathered from visual inspection of reproducible, high-quality 2D-PAGE gels without the assistance of computer programs. The lack of a computer system for analysis should not be a major factor in considering the use of 2D-PAGE. Troubleshooting in 2D-PAGE Because of the complex nature of 2D-PAGE methods, there are technical problems that are frequently encountered while conditions are being optimized for a particular sample. 3,~2,~3A summary of these problems and some suggestions for resolving them are presented in Table I. If the described protocols are followed precisely, only high-quality reagents are used, and care is taken to properly prepare the sample, the 2D-PAGE protein separation and resolution should be excellent. Strategies to Optimize 2D-PAGE Resolution The 2D-PAGE methods described in this chapter are standard procedures widely used by different laboratories that enable the direct compari12 J. VanBlerkom, in "Methods in Mammalian Reproduction" (J. C. Daniel, Jr., ed.), p. 67. Academic Press, New York, 1978. 13 R. Bravo, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 4. Academic Press, New York, 1984.
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TABLE I COMMON PROBLEMSASSOCIATEDWITH 2D-PAGE Problem First dimension Gels do not polymerize
Gels break during focusing
Possible causes
Solutions
Inferior, aged, or improperly prepared catalysts; impure reagents containing contaminants which interfere with polymerization Hole in pH gradient
Start over with fresh, highquality reagents, and check pipetting measurements
Gel is overloaded Gels fall out of tube during focusing Gels will not come out of tubes after focusing Poor separation of proteins
Too much NP-40 in gel
Tubes are not cleaned properly
Improper ampholyte pH range Insufficient protein solubilization Nucleic acid contamination
IEF patterns vary from day to day
Second dimension Irregular gradients
Changing electrofocusing time
Mix ampholytes from two different sources Reduce amount of protein loaded on gel Measure using clipped off pipet tip and wipe outside surface of tip Wash tubes with Chromerge and rinse with water only; do not use methanol or siliconizing solutions Try another pH range Increase ratio of solubilization reagent to protein sample If gels are to be silver stained, increase amount of solubilization reagent and ultracentrifuge at 2 x 105 g. If proteins are to be detected by autoradiography, add nuclease preparations to sample Standardize voltage-hours
Different lots or sources of ampholytes used Stock reagents are too old Inadequate solubilization
Standardize ampholyte source Prepare fresh reagents Increase amount of solubilization reagent
Improper equipment or technique; acrylamide polymerization is too fast
Decrease slightly the amount of catalyst
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TWO-DIMENSIONAL GEL ELECTROPHORESIS TABLE I (continued) Problem
Possible causes
"Fuzzy" protein spots
IEF gel was equilibrated too long before loading onto second dimension gel; not optimal acrylamide concentration in SDS gel Nucleic acid contamination of protein sample Improper or incomplete solubilization
Streaking
Gel is overloaded Sample may contain glycoproteins, which often streak in PAGE
Solutions Use a gradient gel, or vary the acrylamide concentration slightly See First Dimension, poor separation of proteins Increase amount of solubilization reagent, or change type of reagent used Reduce amount of protein on gel Reduce amount of sample on gel, or deglycosylate glycoproteins
son of protein patterns independently generated by individual research groups. It is occasionally necessary, however, to modify these procedures for unusual proteins or for a protein which is extensively posttranslationally modified. F o r example, mixing ampholytes of different ranges may improve resolution of proteins within a particular pH range. Also, protein samples containing an abundance of one protein can alter the p H range of the ampholytes themselves. Therefore, it may be necessary to c o m p e n s a t e by adding different ranges of ampholytes to the isoelectric focusing dimension. When carrying out the initial 2D-PAGE analysis of any protein mixture, it is helpful to include a lane for one-dimensional S D S - P A G E separation of the original sample on the same second dimension polyacrylamide gel. This will determine if any of the protein components are outside the range of the ampholytes used for IEF. Depending on the sample and the complexity of the protein pattern, it may be necessary to try different solubilization conditions, different ampholyte ranges, or N E P H G E (nonequilibrium p H gradient electrophoresis) gels to establish the best method to resolve all the proteins of interest. Standardization of 2 D - P A G E The standardization of 2D-PAGE methods has b e c o m e more important as the need for interlaboratory comparisons of protein patterns has
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IEF 9
4M r x 10 -3
m
t/) 0 =
O I'll
FIG. 1. Example of cellular proteins resolved by high-resolution two-dimensional polyacrylamide gel electrophoresis using color-based silver stain 3,7 illustrating the standardized method for presenting 2D-PAGE protein patterns.
increased. As discussed above, the sophisticated equipment now available for running and analyzing two-dimensional polyacrylamide gels has advanced the standardization process. Another factor enabling accurate comparisons among gels is the use of internal standards for both charge and molecular weight separations. (The procedures which measure pH directly in the gels or in gel slices have proved to be totally inadequate and not reproducible.) Proteins which are modified by carbamylation have proved to be excellent charge standardsy and numerous types of molecular weight standards are commercially available. Publication Format of 2D-PAGE Patterns The protein patterns obtained by 2D-PAGE can be extremely complicated. However, reproducible protein patterns can easily be recognized if they are presented in a standardized format. Many different laboratories, in conjunction with the International Electrophoresis Society, have agreed on a useful presentation format: the acidic end of the IEF gel at the left, and the basic end at the right; i.e., low pH values at the left, increasing toward the right. The second dimension separation is oriented with low-molecular-weight proteins at the bottom and high molecular weights
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at the top, which is standard for 1D-PAGE analysis. The presentation of such a gel is illustrated in Fig. I. This format is now required by the Journal of Electrophoresis and will likely be required by other journals in the future. Acknowledgments The authors wish to acknowledgethe numeroustechniciansand graduate students who have assisted in the developmentof these techniquesover the years. We thank Drs. N. L. Anderson, S. Tollaksen, and D. Sammonsfor manyfruitfuldiscussions,and Ms. Suzanne Mascola for expert secretarial assistance.
[35] I s o e l e c t r i c F o c u s i n g
By DAVID E. GARFIN Proteins, as amphoteric molecules, carry positive, negative, or zero net charges depending on the pH of their local environments. The overall charge of a particular protein is determined by the ionizable acidic and basic side chains of its constituent amino acids and prosthetic groups. Carboxylic acid groups (--COOH) in proteins are uncharged in acidic solutions and dissociate to the anionic form ( - - C O 0 - ) at higher pH values, above about pH 3. Amines (--NH2) and other basic functions of proteins, such as guanidines, are uncharged at alkaline pH, but are cationic below about pH 10 (e.g., --NH3+). The pH at which individual ionizable side chains actually dissociate is affected by the overall composition of the protein and the properties of the medium. As a result, each individual ionizable group in a protein has a nearly unique dissociation point. The net charge on a protein is the algebraic sum of all its positive and negative charges. There is, thus, a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed pl, is a characteristic physicochemical property of every protein. If the number of acidic groups in a protein exceeds the number of basic groups, the pl of that protein will be at a low pH value. If, on the other hand, basic groups outnumber acidic groups, the pI will be high. Proteins show considerable variation in isoelectric points, but pl values usually fall in the range of p H 3 t o p H 10. Proteins are positively charged in solutions at pH values below their pl values and negatively charged above their isoelectric points. In electroMETHODS IN ENZYMOLOGY, VOL. 182
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