[35]
ISOELECTRICFOCUSING
459
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.
460
PURIFICATION PROCEDURES: ELECTROPHORETICMETHODS
[35]
phoresis, the net charge on a protein determines the direction o f its migration (electrophoretic mobility). At p H levels below the p l of a particular protein it will migrate toward the cathode. Conversely, at pH values above its pI a protein will m o v e toward the anode. A protein at its isoelectric point will not migrate in either direction. Isoelectric focusing (IEF) is a technique that was developed from these concepts to separate proteins on the basis of differences in their p l values. It is used for both the analysis and preparative isolation of proteins. I E F , generally carried out under nondenaturing conditions, is a highresolution technique. Resolution of proteins differing in their pI values by only 0.02 p H unit, or less, is common. Because o f this high resolution, protein samples which appear to be homogeneous when tested by other means can often be separated into several components by IEF. Such microheterogeneity may be indicative of differences in primary structure, conformational isomers, differences in the kinds and numbers o f prosthetic groups, or denaturation. The theoretical and practical aspects o f I E F are well documented. Accounts o f all aspects of the field, in more detail than can be presented here, can be found in Refs. 1-9. The methods presented in this chapter are simple, effective, and widely applicable for both analytical and preparative IEF. Principle of Method I E F is an electrophoretic method in which amphoteric molecules are separated as they migrate through a p H gradient. When a protein is placed in a medium with varying pH and subjected to an electric field, it will initially m o v e toward the electrode with the opposite charge. During migration through the p H gradient, the protein will either pick up or lose P. G. Righetti, "Isoelectric Focusing: Theory, Methodology and Applications." Elsevier, Amsterdam, 1983. 2 R. C. Allen, C. A. Saravis, and H. R. Maurer, "Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques." de Gruyter, Berlin, 1984. 3 A. T. Andrews, "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Second Edition." Oxford Univ. Press, New York, 1986. 4 p. G. Righetti, in "Electrokinetic Separation Methods" (P. G. Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 389. Elsevier, Amsterdam, 1979. 5 A. R. Williamson, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd Ed., p. 9.1. Blackwell, Oxford, 1978. 6 B. J. Radola, this series, Vol. 104, p. 256. 7 B. An der Lan and A. Chrambach, in "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. Rickwood, eds.), p. 157. IRL Press, Oxford, 1981. 8 O. Vesterberg, this series, Vol. 22, p. 389. 9 p. G. Righetti, E. Gianazza, and K. Ek, J. Chromatogr. 184, 415 (1980).
[35]
ISOELECTRICFOCUSING
461
protons. As it does, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equaling its pI. There, being uncharged, it will stop migrating. If a protein at its pl should happen to diffuse to a region of lower pH, it will become protonated and be forced toward the cathode by the electric field. If, on the other hand, it diffuses into a pH higher than its pl, the protein will become negatively charged and it will be driven toward the anode. Thus, in this way, proteins condense, or focus, into sharp bands in the pH gradient at their individual, characteristic pl values. Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pl values at differing rates but remain relatively fixed at those pH values for extended periods. This type of motion is in contrast to conventional electrophoresis, in which proteins continue to move through the medium until the electric field is removed. Moreover, in IEF proteins migrate to their steady-state positions from anywhere in the system. This means that, unlike other electrophoretic methods, the sample application point is arbitrary. In fact, the sample can be initially distributed throughout the entire separation system. The key to IEF is the establishment of stable pH gradients in electric fields. This is most commonly accomplished by means of commercially available, synthetic carrier ampholytes (amphoteric electrolytes). These compounds are mixtures of relatively small, multicharged, amphoteric molecules with closely spaced pl values and high conductivity. Under the influence of an electric field, carrier ampholytes partition themselves into smooth pH gradients which increase monotonically from the anode to the cathode. The slope of the pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes. Practical Aspects Format
At one time, all IEF was carried out in vertical columns using density gradients of sucrose or glycerol to stabilize the pH gradient against convection and to support separated zones. ~,3-5,8However, IEF columns are cumbersome and difficult to operate. Focused zones are inherently unstable (because they are denser than the surrounding medium) and not adequately maintained by density gradients. In addition, the resolution obtained by focusing in columns is usually lost during recovery of the focused materials. As a consequence, density gradients have, for the most part, been replaced by other stabilizing media.
462
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
Most analytical IEF is currently carried out in continuous polyacrylamide gels. 1-7 Polyacrylamide provides the virtually uncharged support matrices required for IEF. Gels are formed with large pores which allow the relatively unimpeded motion of proteins. The most common configuration for IEF is the horizontal gel slab. This configuration provides good cooling efficiency and makes sample application relatively easy. Electrofocusing run in cylindrical tubes 7 constitutes the first dimension of the most common two-dimensional gel electrophoresis method. ~° Preparative electrofocusing, too, is a practical reality. Unlike other forms of electrophoresis, the IEF mechanism lends itself to preparative methods. For laboratory-scale protein isolations, density gradients and IEF in beds of granular polyacrylamide or dextran have been used. 14.6.8.11 However, the recently introduced rotating IEF device (the Rotofor cell), 12'~s described below, is becoming the method of choice for laboratory-scale preparative work.
Polyacrylamide Gels Polyacrylamide gels are used for focusing proteins up to about 500,000 Da in size. j-6,t4 They are formed by copolymerization of acrylamide monomer, C H 2 = C H - - C O - - N H 2 , and a cross-linking comonomer, N,N'-methylenebisacrylamide, CHz=CH--CO--NH--CH2--NH-CO--CH~---CH2 (bisacrylamide). Polymerization is through a vinyl addition mechanism catalyzed by a free radical-generating system. In IEF, polymerization is initiated by combined use of ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and illuminated riboflavin. The photochemical initiator, riboflavin, is included because the APSTEMED system is inefficient at low pH. The IEF gel matrix must be both nonsieving and mechanically stable. A suitable gel composition for horizontal electrofocusing is 5% (w/v) total monomer (acrylamide + bisacrylamide) with the cross-linker, bisacrylamide, accounting for 3% (w/w) of the total monomer. By convention, this gel composition is denoted by the pair of figures 5% T, 3% C.
Agarose Gels Agarose gels have much larger pores than polyacrylamide gels. They are used for the separation of large proteins and structures that cannot be ~0 B. S. Dunbar, H. Kimura, and T. M. Timmons, this volume [34]. i~ M. D. Frey and B. J. Radola, Electrophoresis 3, 216 (1982). t2 N. B. Egen, W. Thormann, G. E. Twitty, and M. Bier, in "Electrophoresis '83" (H. Hirai, ed.), p. 547. de Gruyter, Berlin, 1984. is M. Bier, U.S. Pat. 4,588,492 (1986). ~a D. E. Garfin, this volume [33].
[35]
ISOELECTRICFOCUSING
463
readily characterized in polyacrylamide. Molecules larger than 200,000 Da can be separated in 1% agarose gels. The gels are formed by melting an agarose mixture and pouring it onto a glass plate in a manner similar to the methods used in preparing agarose gels for immunoassays and for electrophoresis of DNA. To minimize e lectroendoosmotic solvent flows, only agarose prepared specifically for IEF (zero -Mr) should be used and the viscosity of the medium should be increased by incorporating sorbitol and glycerol into the gels. Consult Refs. 1-3 and 9 for procedures for carrying out agarose IEF.
Apparatus Horizontal slab gels possess a number of advantages and have become very popular for analytical IEF 1-4,9 (preparative devices are discussed below). Gels are cast on glass plates or specially treated plastic sheets and run with one face exposed. This allows samples to be applied anywhere desired on the gel surface, and enables pH and voltage measurements to be made directly on the gel surface. With the slab configuration, a number of samples can be compared under identical running conditions, and most apparatuses allow gel lengths and thicknesses to be varied. Horizontal, flat-bed electrophoresis cells can be obtained from a number of manufacturers. The better quality cells have cooling platforms for heat dissipation, condensation control, and movable electrodes that make direct and uniform contact with the gel surface. Most systems also include devices for casting gels. An alternative arrangement for analytical IEF that has recently been rediscovered is the "inverted" gel format in which the gel is run facing downward suspended between two carbon rod electrodes. 5:5 Inverted cells are less expensive and simpler to use, but less versatile, than standard cells. These cells run at lower voltages than standard fiat beds and require no active cooling. Resolution is somewhat less than can be obtained with standard horizontal cells. The electrical power supply used with standard cells should be capable of delivering up to 3000 V and 30 W operating power. Inverted cells require only about 500 V and 5 W maximum power. Ideally, the power supply will have a constant power mode of operation. Standard cells require coolant circulation for optimum performance. Casting Gels Gels are cast containing carrier ampholytes, pH gradients are established during the runs, concurrently with protein separation. The standard 15Z. L. Awdeh, A. R. Williamson, and B. A. Askonas, Nature (London) 219, 66 (1968).
464
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[3S]
gel thickness of 0.8 or 1 mm provides easily handled gels with good protein load capacity and good staining speed. Thinner gels, 0.2 or 0.4 mm thick, allow higher voltages to be used for increased resolution and shortened run times. Irregularities in gel surfaces and trapped bubbles must be avoided because they cause local distortions in the electric field. Of the various apparatuses available for preparing gels, J-4,9 the capillary method provides the fastest and easiest method for forming gels for IEF.I,2,16,17 A good capillary casting tray IJ6 is a worthwhile investment. These units are acrylic trays with precision spacer rails along their edges for establishing gel thicknesses. Gels are formed by introducing catalyst-activated monomer solution into the space between the acrylic tray and a glass plate or treated plastic sheet and allowing the monomer to polymerize. Since the acrylic surfaces are not wetted by monomer solutions, gels do not stick to the trays. Polyacrylamide gels adhere to the glass (or treated plastic) backing plates and are easily lifted from the trays.
Carrier Ampholytes Carrier ampholytes are complex mixtures of synthetic amphoteric buffers that form smooth pH gradients in applied electric fields. 1-4,6-8j8,~9 Several varieties of carrier ampholytes are commercially available. The products from different manufacturers are not necessarily interchangeable and may yield different IEF patterns. 2 Exact details of the chemical and physical properties of carrier ampholytes are proprietary. In general, they are mixed polymers (about 300-1000 Da in size) of aliphatic amino and carboxylic acids (polyamino-polycarboxylic acids), although some types contain sulfonic and phosphonic acid residues. Following synthesis, carrier ampholytes are purified and blended by the manufacturers to give smooth and reproducible gradients covering wide or narrow pH ranges. Unknown pl values are estimated with wide-range carrier ampholytes covering 7-8 pH units (e.g., pH 3 to I0) and more closely established with narrow pH ranges. The proper choice of ampholyte range is very important to the success of a fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pl of the proteins of interest to ensure that they focus in the linear part of the gradient while excluding extraneous proteins from the separation zone. Moreover, the resolution obtainable in an IEF run depends on the pH profile in the focused gel; 16J. F. Monthony, U.S. Pat. 4,246,222 (1981). 17 R. C. Allen, Electrophoresis 1, 32 (1980). is W. W. Just, this series, Vol. 91, p. 281. 19 S. Binion and L. S. Rodkey, Anal. Biochern. 112, 362 (1981).
[35]
ISOELECTRIC FOCUSING
465
narrow pH range gradients favor high resolution by spreading out the pl values in the region of interest. The range of carrier ampholytes used in an experiment need not be limited to those commercially available. Almost any range desired can be custom made in the laboratory by the methods of preparative IEF. The concentration of carrier ampholytes is also important. Carrier ampholyte concentrations of about 2% (w/v) should be used. Concentrations of ampholytes below 1% (w/v) often result in unstable pH gradients. Above 3% (w/v), ampholytes are difficult to remove from gels and, since they are stainable, they can interfere with protein detection. Resolution
A goal of both analytical and preparative electrophoresis is to achieve the greatest possible degree of resolution between adjacent protein bands. In this context, resolution refers to separation of protein bands relative to their band widths and is denoted by the difference in pI between clearly distinguishable bands. Two of the factors which enter into successful IEF resolution are under direct experimental control. These are the electric field and the steepness of the pH gradient, as determined by the applied voltage and the pH range of the carder ampholytes, respectively. According to both theory and experiment, the difference in pl between two resolved adjacent protein IEF bands (Apl) is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient (field strength) at the position of the bandsl-4,6.8,z0:
ApI ~ (pH gradient/voltage gradient) I/2 Thus, narrow pH ranges and high applied voltages give high resolution (small Apl) in IEF. In addition to these two factors, good resolution is favored by substances with low diffusion coefficients and high rates of change of mobility with pH near their isoelectric points. Most proteins satisfy the latter two criteria, but these factors are, of course, not under the control of the experimenter. Changing the interelectrode distance for a given voltage and pH range will change both the pH and voltage gradients to the same extent, so, unless the carrier ampholyte range or applied voltage is also adjusted accordingly, there will be no alteration in obtainable resolution. In addition to the effect on resolution, high electric fields also result in shortened run times. However, high voltages in electrophoresis are accompanied by large amounts of generated heat (Joule heating). Thus, 20 j. C. Giddings and K. Dahlgren, Sep. Sci. 6, 345 (1971).
466
PURIFICATION PROCEDURES; ELECTROPHORETIC METHODS
[35]
there are limitations on the magnitudes of the electric fields which can be applied. This is partly because resolution decreases with increasing temperature (since diffusion coefficients increase with temperature) and partly because gels can actually get hot enough to burn. Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat than thick ones and are therefore capable of higher resolution. Electric fields used in IEF are generally of the order of 100 V/cm.
Gradient Instability For most practical purposes, pH gradients are stable once the steady state has been reached. However, during extended focusing runs (longer than about 3 hr under standard analytical conditions), gradients are found to slowly deteriorate.l-4,7 This decay is characterized by a drift of the gradients toward the cathode and is accompanied by acidification at the anode, flattening of the gradient in the neutral pH region, and a loss of alkaline bands. The mechanism of the instability, which has been called "cathodic drift," is not completely understood. Cathodic drift is probably caused by a combination of factors including electroendoosmosis, 21 COz absorption, 22 and nonzero, pI-dependent electrophoretic fluxes. 23 The practical consequence of cathodic drift is that excessively long IEF runs should be avoided.
Additives Many protein samples require the use of detergents for their solubilization. For IEF work, the zwitterionic detergents CHAPS and CHAPSO, or the nonionic detergent octylglucoside at concentrations of I-2% in the gel are recommended. Consult Refs. 1-4 and 24 for details. Even in the presence of detergents, some samples may have stringent salt requirements. Only if salt is an absolute requirement should it be present in a sample, and substantial band distortions should be expected. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts. Urea is a common solubilizing agent, especially for those proteins which precipitate at their isoelectric points, even though it denatures proteins.l-4 Urea (3M) is often found satisfactory for maintaining protein 2~ H. Rilbe, in "Electrofocusing and Isotachophoresis" (B. J. Radola and D. Graesslin, eds.), p. 35. de Gruyter, Berlin, 1977. 22 H. Delincre and B. J. Radola, Anal. Biochem. 90, 609 (1978). 23 R. A. Mosher, W. Thormann, and M. Bier, J. Chromatogr. 351, 31 (1986). z4 L. M. Hjelmeland and A. Chrambach, Electrophoresis 2~ 1 (1981).
[35]
ISOELECTRICFOCUSING
467
solubility, but concentrations up to 8 M urea have been used. Only fresh solutions of urea, treated with a mixed bed ion-exchange resin, should be used in order to prevent carbamylation of amine and sulfhydryl groups of proteins. Experimental Procedure The following protocol describes the use of polyacrylamide slabs for IEF on horizontal flat-bed cells. Gel preparation, sample application, focusing conditions, and detection methods are included. The gel recipe is for 12 ml of 5% T (3% C) acrylamide, 2% ampholytes, and 5% glycerol. This is sufficient for casting one standard-size gel of 100 x 125 × 0.8 mm (10 ml) or four 100 × 125 × 0.2 mm gels (10 ml total). The thinner gels can be run at twice the voltage of the thicker ones for increased resolution. Equipment and reagents for IEF are available from many suppliers. For best results, follow the manufacturer's instructions and recommendations, especially when working with high-voltage equipment. Except where noted, reagents for IEF can be prepared as concentrated stock solutions. All water used should be distilled or deionized. Stock Solutions Acrylamide monomer concentrate (25% T, 3% C): Dissolve 24.25 g acrylamide and 0.75 g bisacrylamide in about 70 ml of water. Adjust the final volume to 100 ml. Filter through a 0.45-ttm filter. Store protected from light at 4 ° for up to 1 month. Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, do not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder or solutions containing it. For disposal of unused acrylamide, add bisacrylamide (if none is present), induce polymerization, and discard the solidified gel 25% glycerol (w/v): Weigh 25 g glycerol in a beaker. Add about 50 ml of water and mix well. Dilute to 100 ml with water Carrier ampholytes: Use ampholytes undiluted unless instructed otherwise by the manufacturer. Carrier ampholytes are supplied as aqueous solutions, usually containing 40 or 20% (w/v) solids. The pH range used will depend on the protein(s) of interest 0.1% (w/v) riboflavin 5'-phosphate (FMN): Dissolve 50 mg riboflavin 5'-phosphate in 50 ml water. Store protected from light at 4 ° for up to 1 month 10% (w/v) ammonium persulfate (APS): Dissolve 100 mg APS in 1 ml of water. Prepare this solution fresh daily
468
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
TEMED (N,N,N',N'-tetramethylethylenediamine): Use TEMED undiluted as supplied. Use only pure, distilled TEMED. Store cool and protected from light
Casting Gels The use of gel support film for polyacrylamide is highly recommended, especially with thin gels, which cannot be easily handled unless supported. Polyacrylamide binds covalently to these sheets of treated polyester, 2,25 simplifying gel handling in all steps, from running gels through drying and storing them. Although polyacrylamide gels adhere to wellcleaned glass plates and remain bound through the IEF runs, gels will come off of the backing plates during the staining or destaining steps. IEF gels are very difficult to manipulate once they become detached from their backings. Basic ampholytes (pH > 8) may interfere with the adhesion of gels to support films. Increasing the APS concentration in the final gel to 0.7 mg/ml (84/xl of 10% APS/12 ml of the gel solution given below) should alleviate the problem. Prolonged soaking in the acidic staining and destaining solutions can also affect adhesion of polyacrylamide gels to the support films. Do not soak the gels any longer than necessary in the staining and destaining solutions. 1. Place a few drops of water on a clean glass IEF plate and place the hydrophobic side of a gel support film against the plate (water beads on the hydrophobic sides of the films). Roll the support film fiat with a test tube or similar object to force out excess water and air bubbles and wipe off excess liquid at the edges. Capillarity is sufficient to hold the supported gel on the plate throughout the run. Place the glass plate on the casting tray with the gel support film facing down. 2. Prepare monomer-ampholyte solution from the stock reagents: Water Monomer concentrate 25% (w/v) glycerol 40% ampholyte (w/v)
6.6 2.4 2.4 0.6
ml ml ml ml
Adjust the volume of water to accommodate additives and different ampholyte concentrations; e.g., with 20% ampholytes use 6 ml of water and 1.2 ml of ampholyte solution. 3. Deaerate the monomer-ampholyte solution under vacuum for 5 min; e.g., in a bell jar, desiccator, or vacuum flask. 25 B. J. Radola,
Electrophoresis 1,
43 (1980).
[35]
ISOELECTRICFOCUSING
469
4. Add initiators, swirling gently to mix them into the monomerampholyte solution. 0.1% (w/v) FMN 10% (w/v) APS TEMED (undiluted)
60/zl 18 tzl 4/zl
5. Using a pipet and bulb, carefully introduce the monomer solution between the support film-glass plate and the casting tray. Control the flow to prevent air bubbles. If a bubble becomes trapped in the monomer solution, slide the plate sideways until the bubble escapes at an edge, then reposition the plate so that there is a uniform layer of monomer under it. 6. Position a fluorescent lamp directly over the tray about 3-4 cm from the gel. Illuminate the solution for about 45 min. 7. Lift the gel from the tray by gently prying it up with a spatula. Turn the plate over, with the gel upward, and illuminate the gel for a further 20 min to polymerize monomer remaining on the gel surface. 8. The gel may be used immediately or it can be covered in plastic wrap and stored at 4 ° for several days. Best results are sometimes obtained by letting a gel " c u r e " (polymerize completely) overnight at 4 ° before use.
Sample Preparation Protein samples for IEF must be substantially salt free and free of precipitates. Small samples (1 to 10 tzl) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable sample solutions can be prepared by dialysis or gel filtration. Good visualization of focused lanes generally requires a minimum of 0.5 /xg of protein/band with dye staining or 50 ng of protein/band with silver staining (see below).
Sample Application There are many suitable methods for applying samples to thin-layer polyacrylamide gels. ~-4One of the simplest methods is to place filter paper strips impregnated with sample directly on the gel surface. Up to 25 tzl of sample solution can be conveniently applied after absorption into 1-cm squares of filter paper. A convenient size for applicator papers is 0.2 x 1 cm, holding 5/zl of sample solution. There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH
470
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
the samples should not be applied closer than 1 cm from either electrode. Preforming the pH gradient before sample application will also limit the exposure of proteins to pH extremes. Some polypeptides are eluted most efficiently from strips when applied to the anodic side of the ge1.1,4,26 A good strategy when focusing a protein for the first time is to apply samples to three different areas of the gel, one near each electrode and one near the middle of the gel. This strategy is also valuable in estimating the approach to steady-state focusing. 3,22 When the patterns obtained on applying the sample at opposite ends of the gel become identical, the steady state can be assumed to have been reached. Steady-state conditions should be duplicated when determining the pl values of particular proteins. It should be noted that samples applied at opposite ends of IEF gels will not always focus into identical patterns. The reason for this is not clear, but may be related to interactions of proteins with differing species of carrier ampholytes during focusing.
Focusing 1. Connect theelectrophoresis cell to a refrigerated circulator cooled to 4 °. 2. Wet the cooling stage with a few drops of water or 0.5% glycerol to ensure good thermal contact with the gel backing. Place the gel on the platform (gel upward). Blot any excess liquid from the cooling stage to eliminate possible electrical shorting paths. 3. Cut electrode strips. Thick filter paper or (uncolored) blotter paper work well as electrode strips. More than one thickness can be used. The strips should be about 7 mm wide and cut about 4 mm shorter than the width of the gel (this prevents electrical arcing to the cooling stage). Place the electrode strips on a glass plate and wet them with the appropriate electrolyte solutions. The anode solution is acidic and the cathode solution is basic. a. For most purposes, use 1 N NaOH as catholyte (negative terminal) and 1 N H3PO4 as anolyte (positive terminal).1-4 b. At high voltages, as in focusing with ultrathin gels (0.2 mm or less), the following electrolytes are recommended25: Catholyte: 20 mM lysine, 20 mM arginine, 2 M ethylenediamine. Dissolve 0.36 g lysine (free base), 0.34 g arginine (free base), and 13.4 ml ethylenediamine in water to give 100 ml. Two molar ethanolamine (12 ml/100 ml) can be substituted for the ethylenediamine. Store at 4 °. 26 p. G. Righetti and F. Chillemi, J. Chromatogr. 157, 243 (1978).
[35]
ISOELECTRIC FOCUSING
471
Anolyte: 20 mM aspartic acid, 20 mM glutamic acid. Dissolve 0.26 g aspartic acid and 0.29 g glutamic acid in water to give 100 ml of solution. Store at 4° . 4. Blot the wetted electrode strips with paper towels until they are slightly moist. Carefully place the strips along the appropriate edges of the gel. Do not allow the strips to extend beyond the sides of the gel. 5. Cut 0.2 × 1 cm pieces of filter paper for sample application. Place the pieces of paper on a glass plate and pipette 5/~l of a protein sample solution to each piece. Place the applicator papers on the gel l cm from the anode (sample application is discussed above). 6. Position the electrodes over the electrolyte strips, making sure that there is good contact between the electrodes and the strips. 7. Set the power supply to constant power and run at 6 W/gel, with a voltage limit of 1500 V for standard size gels (100 x 125 x 0.8 mm). Total run time is about 2 hr. The actual running conditions will vary with the apparatus, the gel thickness, the sample solution, and the ampholytes. Gels should always be run at the highest voltage compatible with the heat-dissipation capabilities of the electrophoresis cell. Check the manufacturer's recommendations for proper power settings. At the start of a run, when voltage is first applied, the current will be at its highest value, because the carrier ampholytes have not yet focused. As the run progresses, the conductivity of the gel will drop and the current will fall. It is recommended that the run be started in the constant power mode set at the heat-dissipation limit of the cell. The power supply should be maintained in this mode until the current drops to its lowest value and the highest voltage is reached. (Recall that power = voltage x current and current = voltage x conductivity.) Standard-size gels run at 6 W usually plateau at 1200-1500 V in about l hr. When the maximum voltage is reached, switch to constant voltage regulation (at the voltage limit) for the remainder of the focusing run. The paper application strips can be removed at the time the switch to the constant voltage mode is made. A good way to monitor the progress of a run is with colored marker proteins. However, not all proteins focus at the same rate, so that for accuracy and reproducibility, it is necessary to predetermine the correct conditions for reaching the steady state for the protein of interest. Initial power settings of l0 W/100 × 125 mm gel are common, and final voltage gradients as high as 300 V/cm interelectrode distance have been used with thin gels (0.2 mm). It is customary to characterize the extent of focusing in IEF runs with the time integral of the applied voltage, expressed in volt-hours. 2,22 The volt-hour designation is meant as a standard for reproducing focusing
472
PURIFICATION PROCEDURES; ELECTROPHORETIC METHODS
[35]
conditions. The conditions for attaining steady-state focusing, once determined, are reproducible. However, many factors, especially temperature, affect the absolute reproducibility of focusing. Thus, although the volthour quantity is a convenient indicator of the extent of focusing, it is not a definitive measure of the IEF process. High voltages run for short times result in better separations than low voltages and long times. Detection of Protein Bands Protein staining is the most general method of detection. Discussions of other detection methods and means for quantitating protein bands in gels can be found in Refs. 1-4. Carry out staining and destaining steps at room temperature with gentle agitation (e.g., on an orbital shaker platform) in any convenient container, such as a glass casserole or photography tray. Carefully peel off sample paper strips and electrode wicks (if possible) before beginning the procedure. Standard Procedure. No preliminary fixation step is required with this method. Bands containing proteins in microgram quantities are easily seen. 1. Prepare the staining solution: 0.04% Coomassie Brilliant Blue R-250, 0.05% Crocein Scarlet, 0.5% CuSO4 in 27% ethanol, 10% acetic acid: Water CuSO4 Ethanol Glacial acetic acid Coomassie Brilliant Blue R-250 Crocein Scarlet
630 5.0 270 100 0.4 0.5
ml g ml ml g g
2-Propanol can be substituted for ethanol. Dissolve the cupric sulfate in the water before adding the alcohol. Add the dyes to the solution last. Filter the solution after the dyes have dissolved. The staining solution is reusable. Store it at room temperature. Crocein Scarlet rapidly binds and fixes proteins. 27 Cupric sulfate enhances stain intensity. 1,4,28 2. Soak gels in staining solution for at least 1 hr. 3. Destain with a large excess of 12% ethanol. 7% acetic acid, 0.5% CuSO4 (810 ml H20, 5 g CuSO4, 120 ml ethanol, 70 ml acetic acid) until a clear background is obtained. This will require several changes of destaining solution. Grainy precipitates of dye will sometimes settle on the surfaces of gels after the staining solution has been reused several times. 27 A. J. Crowle and L. J. Cline, J. lmmunol. Methods 17, 379 (1977). 28 p. G. Righetti and J. W. Drysdale, J. Chromatogr. 98, 271 (1974).
[35]
ISOELECTRICFOCUSING
473
These precipitates can be wiped off of the gel with a gloved finger or a moistened tissue while the gel is in the destaining solution. 4. Soak the gel in 12% ethanol, 7% acetic acid to remove the cupric sulfate. Crocein Scarlet can be omitted from the staining solution. If so, gels must be immersed in fixative (4% sulfosalicylic acid, 12.5% trichloroacetic acid, 30% methanol) for at least 30 min prior to staining. If ultra-thin (0.2 mm) gels detach from gel support film during the staining procedure, shorten immersion times, so that the total staining and destaining takes no more than 2 hr. Quick Stain. The following technique 29 is nearly as sensitive as the above one and requires no destaining (it cannot be used in the presence of detergents): Immerse the gel for 1 hr in 3.5% perchloric acid containing 0.025% Coomassie Brilliant Blue G-250. For intensification, immerse the gel in 7% acetic acid. Silver Stain. Silver staining is 10 to 100 times more sensitive than dye staining. Before beginning the silver staining procedure, IEF gels must first be fixed in 30% methanol, 10% trichloroacetic acid, 3.5% sulfosalicylic acid for 1 hr, followed by at least 2 hr in several volumes of 30% methanol, 12% trichloroacetic acid. The Merril silver staining protocol 2,14,30 must be modified for IEF gels bonded to gel support film to include two soaks for 5 min each in 400 ml deionized water between the oxidizer and silver reagent steps, and a 1-min wash in 400 ml of water between the silver reagent and developer steps.
Preservation of Gels To dry gels on support film, simply allow them to dry overnight in air in a dust-free location. Alternatively, gels can be carefully dried with a heat gun at a low setting. To dry unsupported gels, first soak them in 7% acetic acid, 5% glycerol for 1 hr, then smooth them on water-wetted filter paper and dry them in a gel dryer.
Determining pH Gradients When focusing is completed, pH gradients can be determined in various ways. The most straightforward method is to base pH profiles on the 29 A. H. Reisner, P. Nemes, and C. Bucholtz, Anal. Biochem. 64, 509 (1975); see also A. H. Reisner, this series, Vol. 104, p. 439. 30 C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Science 211, 1437 (1981); see also C. R. Merril, D. Goldman, and M. L. Van Keuren, this series, Vol. 104, p. 441; C. R. Merril, this volume [36].
474
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
positions of focused marker proteins. Extensive, but outdated, tables of protein isoelectric p o i n t s 31-33 c a n be consulted in choosing appropriate markers for a particular experiment. It is much simpler, though, to make use of one of the many commercially available protein mixtures. 1EF protein standards are combinations of proteins with well-characterized pl values blended to give uniform staining. The blends often contain naturally colored proteins which allow focusing runs to be continually monitored. They also usually contain proteins that achieve steady-state focusing in relatively short times. Marker proteins usually reach the steady state in about 2500 V-hr. Gels are calibrated with one or two lanes of IEF protein standards. Unknown isoelectric points can be interpolated from graphs of the positions of focused marker proteins plotted as functions of their isoelectric points. pH gradients can also be directly determined with surface electrodes or by elution of ampholytes (before staining). In the latter method, first either slice gels with a blade or punch them into closely spaced pieces with a sharp cork borer. Individually soak each piece of gel in a minimum volume of degassed water or 10 mM KCI for 1-2 hr and measure the pH of each solution. Regardless of the method used in determining the gradient, what is actually measured is the pH of the focused carrier ampholytes, not the proteins themselves. Ideally, pH measurements should be made at the same temperature as the IEF run. Nevertheless, temperature and solvent effects and interference from absorption of atmospheric CO2 are usually neglected in most pH determinations unless accurate pl measurements are required. Discussions of the effects of these factors in Refs. 1, 3, 4, 7, and 22 should be consulted. Microheterogeneity and Artifacts. Multiband IEF patterns can arise from molecular interactions and conformation changes as well as from inherent isoelectric microheterogeneity. 34 Ampholytes can reversibly bind directly to proteins, proteins can undergo sequential pH-dependent conformational changes, and proteins can interact with one another. These types of reactions can artifactually alter the pI profiles o f proteins. On the other hand, many proteins are inherently heterogeneous, consisting of isoelectric isomers. To distinguish between artifactual and inherent heterogeneity, single focused bands should be cut out and rerun. If a 31 p. G. Righetti and T. Caravaggio, J. Chromatogr. 127, 1 (1976). 32 D. Malamud and J. W. Drysdale, Anal. Biochem. 86, 620 (1978). 33 p. G. Righetti and G. Tudor, J. Chromatogr. 220, 115 (1981). J. R. Cann, in "Electrokinetic Separation Methods" (P. G. Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 369. Elsevier, Amsterdam, 1979; see also J. R. Cann, this series, Vol. 61, p. 142.
[35]
ISOELECTRICFOCUSING
475
single band splits into multiple bands on refocusing, artifact formation is indicated. When rerunning a band, care should be taken to rerun it under the same conditions and from the same position on the gel as the initial sample. Preparative Isoelectric Focusing Two techniques which are useful for laboratory-scale preparative electrofocusing are IEF in granular beds I-4,6,11,35 and use of the Rotofor cell. 12,~3,36Both methods allow preparative fractionations on the scale of from hundreds of milligrams to grams of protein, with recoveries of greater than 90% possible. 11 Purification levels between 10- and 100-fold place IEF methods intermediate between ion-exchange and ligand-binding chromatographies as preparative methods. IEF is well suited for use at any stage of a preparative scheme, and is particularly effective in the early stages of purification. In many cases, simple sequential fractionation and refractionation on the same device provides the desired purity. It is not necessary to attain steady-state focusing in preparative IEF, since adequate separations may be achieved before then. IEF in Granulated Gel Beds
Focusing in beds of granulated polyacrylamide or dextran allows highresolution separation and recovery of relatively large quantities of protein. ,-4.6,11,35Granular polyacrylamide gels are recommended for this procedure because of low residual charge and resistance to enzymatic degradation. 11Focusing is carried out in ampholyte-containing gel slurries in specially designed trays in standard fiat-bed apparatus. The manufacturer's instructions should be followed for proper use of each particular cell. The Rotofor Cell
The easiest to use preparative electrophoresis device is the Rotofor cell developed by Egen, Bier, and associates 1z,13(available only from BioRad Laboratories). The principle of the Rotofor cell, IEF in free solution, is similar to that of column methods. However, zone stabilization in the Rotofor cell is achieved not by means of density gradients, but by turning the column on its side. Gravitationally induced convection is inhibited by rotating the column about its (horizontal) axis. The separation column is 35 C. Demeulemester, G. Peltre, D. Panheleux, and B. David, Electrophoresis 7, 518 (1986). 36 N. B. Egen, M. Bliss, M. Mayersohn, S. M. Owens, L. Arnold, and M. Bier, Anal. Biochem. 172, 488 (1988).
476
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
divided into compartments by means of screens of woven polyester. The screens offer resistance to fluid convection, but do not hinder the flow of current or the transport of proteins. Proteins, which are initially dispersed uniformly throughout the chamber, migrate to the one or more compartments which are at pH values nearest to their isoelectric points. The combined effect of compartmentalization and rotation is superior to either method alone in maintaining the stability of focused zones. The segmentation of the column also facilitates fraction collection. A focusing chamber, capable of holding up to 55 ml of sample, is divided into 20 compartments by a core made up of 19 disks of polyester screen (6-/~m pores). A ceramic cooling finger runs through the center of the focusing chamber to dissipate the heat generated during the run. Two electrode assemblies hold the anolyte and catholyte solutions. Appropriate ion-exchange membranes and gaskets isolate the electrolytes from the sample in the focusing chamber while allowing electrical contact with the material in the chamber. The anolyte is usually 0.1 M H3PO4 and the catholyte is usually 0.1 M NaOH, but any other electrolytes which are compatible with IEF can be used. Vent caps provide pressure relief from the gases which build up in the electrode chambers by electrolysis during the run. The entire assembly rotates around the central horizontal axis during the run to inhibit convection, maintain even cooling and efficient electrical contact, and prevent the screens from becoming clogged by precipitated protein. Runs are at 4 ° at constant power (12 W) for 4 hr. Simple and rapid sample collection is by aspiration through tubing lines connecting the 20 individual compartments with corresponding test tubes in a vacuum chamber. Collection is accomplished in seconds, minimizing remixing of fractions by diffusion. Some remixing of adjacent zones takes place, however, because of the finite dimensions of the fraction compartments. The individual test tube fractions are easily sampled for assay or measured for pH with standard electrodes. Samples for the Rotofor need not be completely desalted before fractionation. Ions in the sample solution will be electrophoresed into the two end compartments in the early stages of the run. Carrier ampholyte (2%, w/v) in the initial sample solution supplies enough ampholyte for refractionation of pooled material. After the tub~s containing the protein of interest have been identified, the assay peak can be pooled for a second run. The amount of carrier ampholytes contained in the pooled fractions is adequate for refractionation. The pH range covered on refractionation is determined by the pooled fractions and is generally much narrower than the initial range. Twenty-five-fold purification in a single run and 1000-fold purification by refractionation have been achieved. The ideal sample run on the Rotofor cell would contain only the pro-
[36]
GEL-STAININGTECHNIQUES
477
tein mixture, water, and ampholytes. However, pI precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°. Detergents (1-2%, w/v) may also be added to samples. Zwitterionic detergents such as CHAPS, CHAPSO, and nonionic octylglucoside are satisfactory. Triton X-100 and NP-40 may be less satisfactory due to their slight charge content.
Removal of Ampholytes from Proteins There are a number of ways to separate ampholytes from proteins.l-4 Electrophoresis, ammonium sulfate precipitation, and gel filtration, ionexchange, and hydroxylapatite chromatographies have all been used. Dialysis is a simple and effective method for removing ampholytes from solutions of proteins. First, adjust the pooled fractions to 1 M NaCi to disrupt weak electrostatic complexes between ampholytes and proteins, then dialyze the solutions into appropriate buffers. Extensive dialysis is required for thorough removal of ampholytes. There is no good way to demonstrate complete absence of ampholytes in a protein solution, but for many applications they need not be removed.
[36] G e l - S t a i n i n g T e c h n i q u e s
By CARL R. MERRIL Protein Stains Naturally colored proteins such as myoglobin, hemoglobin, ferritin, and cytochrome ¢ may be directly observed in gels illuminated with light in the visual spectrum, providing that their chromophores are not damaged during electrophoresis. 1However, the visualization of most proteins requires the use of dyes or stains. Organic stains were first utilized for the detection of proteins on gels. Recently metal-based stains, such as the silver stains, have achieved widespread use because of their increased sensitivity. A number of organic stains have been adapted for the detection of electrophoretically separated proteins, including Bromphenol Blue, 2 Fast Green (Food Green 3) and Amido Black (Acid Black 1). 3 Some of these 1 B. D, Davis and E. J. Cohn, Ann. N.Y. Acad. Sci. 39, 209 (1939). 2 E. L, Durrum, J. Am. Chem. Soc. 72, 2943 (1950). W. Grassman and K. Hannig, Z. Physiol. Chem. 290, 1 (1952).
METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
ISOELECTRICFOCUSING
459
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.
460
PURIFICATION PROCEDURES: ELECTROPHORETICMETHODS
[35]
phoresis, the net charge on a protein determines the direction o f its migration (electrophoretic mobility). At p H levels below the p l of a particular protein it will migrate toward the cathode. Conversely, at pH values above its pI a protein will m o v e toward the anode. A protein at its isoelectric point will not migrate in either direction. Isoelectric focusing (IEF) is a technique that was developed from these concepts to separate proteins on the basis of differences in their p l values. It is used for both the analysis and preparative isolation of proteins. I E F , generally carried out under nondenaturing conditions, is a highresolution technique. Resolution of proteins differing in their pI values by only 0.02 p H unit, or less, is common. Because o f this high resolution, protein samples which appear to be homogeneous when tested by other means can often be separated into several components by IEF. Such microheterogeneity may be indicative of differences in primary structure, conformational isomers, differences in the kinds and numbers o f prosthetic groups, or denaturation. The theoretical and practical aspects o f I E F are well documented. Accounts o f all aspects of the field, in more detail than can be presented here, can be found in Refs. 1-9. The methods presented in this chapter are simple, effective, and widely applicable for both analytical and preparative IEF. Principle of Method I E F is an electrophoretic method in which amphoteric molecules are separated as they migrate through a p H gradient. When a protein is placed in a medium with varying pH and subjected to an electric field, it will initially m o v e toward the electrode with the opposite charge. During migration through the p H gradient, the protein will either pick up or lose P. G. Righetti, "Isoelectric Focusing: Theory, Methodology and Applications." Elsevier, Amsterdam, 1983. 2 R. C. Allen, C. A. Saravis, and H. R. Maurer, "Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques." de Gruyter, Berlin, 1984. 3 A. T. Andrews, "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Second Edition." Oxford Univ. Press, New York, 1986. 4 p. G. Righetti, in "Electrokinetic Separation Methods" (P. G. Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 389. Elsevier, Amsterdam, 1979. 5 A. R. Williamson, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd Ed., p. 9.1. Blackwell, Oxford, 1978. 6 B. J. Radola, this series, Vol. 104, p. 256. 7 B. An der Lan and A. Chrambach, in "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. Rickwood, eds.), p. 157. IRL Press, Oxford, 1981. 8 O. Vesterberg, this series, Vol. 22, p. 389. 9 p. G. Righetti, E. Gianazza, and K. Ek, J. Chromatogr. 184, 415 (1980).
[35]
ISOELECTRICFOCUSING
461
protons. As it does, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equaling its pI. There, being uncharged, it will stop migrating. If a protein at its pl should happen to diffuse to a region of lower pH, it will become protonated and be forced toward the cathode by the electric field. If, on the other hand, it diffuses into a pH higher than its pl, the protein will become negatively charged and it will be driven toward the anode. Thus, in this way, proteins condense, or focus, into sharp bands in the pH gradient at their individual, characteristic pl values. Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pl values at differing rates but remain relatively fixed at those pH values for extended periods. This type of motion is in contrast to conventional electrophoresis, in which proteins continue to move through the medium until the electric field is removed. Moreover, in IEF proteins migrate to their steady-state positions from anywhere in the system. This means that, unlike other electrophoretic methods, the sample application point is arbitrary. In fact, the sample can be initially distributed throughout the entire separation system. The key to IEF is the establishment of stable pH gradients in electric fields. This is most commonly accomplished by means of commercially available, synthetic carrier ampholytes (amphoteric electrolytes). These compounds are mixtures of relatively small, multicharged, amphoteric molecules with closely spaced pl values and high conductivity. Under the influence of an electric field, carrier ampholytes partition themselves into smooth pH gradients which increase monotonically from the anode to the cathode. The slope of the pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes. Practical Aspects Format
At one time, all IEF was carried out in vertical columns using density gradients of sucrose or glycerol to stabilize the pH gradient against convection and to support separated zones. ~,3-5,8However, IEF columns are cumbersome and difficult to operate. Focused zones are inherently unstable (because they are denser than the surrounding medium) and not adequately maintained by density gradients. In addition, the resolution obtained by focusing in columns is usually lost during recovery of the focused materials. As a consequence, density gradients have, for the most part, been replaced by other stabilizing media.
462
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
Most analytical IEF is currently carried out in continuous polyacrylamide gels. 1-7 Polyacrylamide provides the virtually uncharged support matrices required for IEF. Gels are formed with large pores which allow the relatively unimpeded motion of proteins. The most common configuration for IEF is the horizontal gel slab. This configuration provides good cooling efficiency and makes sample application relatively easy. Electrofocusing run in cylindrical tubes 7 constitutes the first dimension of the most common two-dimensional gel electrophoresis method. ~° Preparative electrofocusing, too, is a practical reality. Unlike other forms of electrophoresis, the IEF mechanism lends itself to preparative methods. For laboratory-scale protein isolations, density gradients and IEF in beds of granular polyacrylamide or dextran have been used. 14.6.8.11 However, the recently introduced rotating IEF device (the Rotofor cell), 12'~s described below, is becoming the method of choice for laboratory-scale preparative work.
Polyacrylamide Gels Polyacrylamide gels are used for focusing proteins up to about 500,000 Da in size. j-6,t4 They are formed by copolymerization of acrylamide monomer, C H 2 = C H - - C O - - N H 2 , and a cross-linking comonomer, N,N'-methylenebisacrylamide, CHz=CH--CO--NH--CH2--NH-CO--CH~---CH2 (bisacrylamide). Polymerization is through a vinyl addition mechanism catalyzed by a free radical-generating system. In IEF, polymerization is initiated by combined use of ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and illuminated riboflavin. The photochemical initiator, riboflavin, is included because the APSTEMED system is inefficient at low pH. The IEF gel matrix must be both nonsieving and mechanically stable. A suitable gel composition for horizontal electrofocusing is 5% (w/v) total monomer (acrylamide + bisacrylamide) with the cross-linker, bisacrylamide, accounting for 3% (w/w) of the total monomer. By convention, this gel composition is denoted by the pair of figures 5% T, 3% C.
Agarose Gels Agarose gels have much larger pores than polyacrylamide gels. They are used for the separation of large proteins and structures that cannot be ~0 B. S. Dunbar, H. Kimura, and T. M. Timmons, this volume [34]. i~ M. D. Frey and B. J. Radola, Electrophoresis 3, 216 (1982). t2 N. B. Egen, W. Thormann, G. E. Twitty, and M. Bier, in "Electrophoresis '83" (H. Hirai, ed.), p. 547. de Gruyter, Berlin, 1984. is M. Bier, U.S. Pat. 4,588,492 (1986). ~a D. E. Garfin, this volume [33].
[35]
ISOELECTRICFOCUSING
463
readily characterized in polyacrylamide. Molecules larger than 200,000 Da can be separated in 1% agarose gels. The gels are formed by melting an agarose mixture and pouring it onto a glass plate in a manner similar to the methods used in preparing agarose gels for immunoassays and for electrophoresis of DNA. To minimize e lectroendoosmotic solvent flows, only agarose prepared specifically for IEF (zero -Mr) should be used and the viscosity of the medium should be increased by incorporating sorbitol and glycerol into the gels. Consult Refs. 1-3 and 9 for procedures for carrying out agarose IEF.
Apparatus Horizontal slab gels possess a number of advantages and have become very popular for analytical IEF 1-4,9 (preparative devices are discussed below). Gels are cast on glass plates or specially treated plastic sheets and run with one face exposed. This allows samples to be applied anywhere desired on the gel surface, and enables pH and voltage measurements to be made directly on the gel surface. With the slab configuration, a number of samples can be compared under identical running conditions, and most apparatuses allow gel lengths and thicknesses to be varied. Horizontal, flat-bed electrophoresis cells can be obtained from a number of manufacturers. The better quality cells have cooling platforms for heat dissipation, condensation control, and movable electrodes that make direct and uniform contact with the gel surface. Most systems also include devices for casting gels. An alternative arrangement for analytical IEF that has recently been rediscovered is the "inverted" gel format in which the gel is run facing downward suspended between two carbon rod electrodes. 5:5 Inverted cells are less expensive and simpler to use, but less versatile, than standard cells. These cells run at lower voltages than standard fiat beds and require no active cooling. Resolution is somewhat less than can be obtained with standard horizontal cells. The electrical power supply used with standard cells should be capable of delivering up to 3000 V and 30 W operating power. Inverted cells require only about 500 V and 5 W maximum power. Ideally, the power supply will have a constant power mode of operation. Standard cells require coolant circulation for optimum performance. Casting Gels Gels are cast containing carrier ampholytes, pH gradients are established during the runs, concurrently with protein separation. The standard 15Z. L. Awdeh, A. R. Williamson, and B. A. Askonas, Nature (London) 219, 66 (1968).
464
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[3S]
gel thickness of 0.8 or 1 mm provides easily handled gels with good protein load capacity and good staining speed. Thinner gels, 0.2 or 0.4 mm thick, allow higher voltages to be used for increased resolution and shortened run times. Irregularities in gel surfaces and trapped bubbles must be avoided because they cause local distortions in the electric field. Of the various apparatuses available for preparing gels, J-4,9 the capillary method provides the fastest and easiest method for forming gels for IEF.I,2,16,17 A good capillary casting tray IJ6 is a worthwhile investment. These units are acrylic trays with precision spacer rails along their edges for establishing gel thicknesses. Gels are formed by introducing catalyst-activated monomer solution into the space between the acrylic tray and a glass plate or treated plastic sheet and allowing the monomer to polymerize. Since the acrylic surfaces are not wetted by monomer solutions, gels do not stick to the trays. Polyacrylamide gels adhere to the glass (or treated plastic) backing plates and are easily lifted from the trays.
Carrier Ampholytes Carrier ampholytes are complex mixtures of synthetic amphoteric buffers that form smooth pH gradients in applied electric fields. 1-4,6-8j8,~9 Several varieties of carrier ampholytes are commercially available. The products from different manufacturers are not necessarily interchangeable and may yield different IEF patterns. 2 Exact details of the chemical and physical properties of carrier ampholytes are proprietary. In general, they are mixed polymers (about 300-1000 Da in size) of aliphatic amino and carboxylic acids (polyamino-polycarboxylic acids), although some types contain sulfonic and phosphonic acid residues. Following synthesis, carrier ampholytes are purified and blended by the manufacturers to give smooth and reproducible gradients covering wide or narrow pH ranges. Unknown pl values are estimated with wide-range carrier ampholytes covering 7-8 pH units (e.g., pH 3 to I0) and more closely established with narrow pH ranges. The proper choice of ampholyte range is very important to the success of a fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pl of the proteins of interest to ensure that they focus in the linear part of the gradient while excluding extraneous proteins from the separation zone. Moreover, the resolution obtainable in an IEF run depends on the pH profile in the focused gel; 16J. F. Monthony, U.S. Pat. 4,246,222 (1981). 17 R. C. Allen, Electrophoresis 1, 32 (1980). is W. W. Just, this series, Vol. 91, p. 281. 19 S. Binion and L. S. Rodkey, Anal. Biochern. 112, 362 (1981).
[35]
ISOELECTRIC FOCUSING
465
narrow pH range gradients favor high resolution by spreading out the pl values in the region of interest. The range of carrier ampholytes used in an experiment need not be limited to those commercially available. Almost any range desired can be custom made in the laboratory by the methods of preparative IEF. The concentration of carrier ampholytes is also important. Carrier ampholyte concentrations of about 2% (w/v) should be used. Concentrations of ampholytes below 1% (w/v) often result in unstable pH gradients. Above 3% (w/v), ampholytes are difficult to remove from gels and, since they are stainable, they can interfere with protein detection. Resolution
A goal of both analytical and preparative electrophoresis is to achieve the greatest possible degree of resolution between adjacent protein bands. In this context, resolution refers to separation of protein bands relative to their band widths and is denoted by the difference in pI between clearly distinguishable bands. Two of the factors which enter into successful IEF resolution are under direct experimental control. These are the electric field and the steepness of the pH gradient, as determined by the applied voltage and the pH range of the carder ampholytes, respectively. According to both theory and experiment, the difference in pl between two resolved adjacent protein IEF bands (Apl) is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient (field strength) at the position of the bandsl-4,6.8,z0:
ApI ~ (pH gradient/voltage gradient) I/2 Thus, narrow pH ranges and high applied voltages give high resolution (small Apl) in IEF. In addition to these two factors, good resolution is favored by substances with low diffusion coefficients and high rates of change of mobility with pH near their isoelectric points. Most proteins satisfy the latter two criteria, but these factors are, of course, not under the control of the experimenter. Changing the interelectrode distance for a given voltage and pH range will change both the pH and voltage gradients to the same extent, so, unless the carrier ampholyte range or applied voltage is also adjusted accordingly, there will be no alteration in obtainable resolution. In addition to the effect on resolution, high electric fields also result in shortened run times. However, high voltages in electrophoresis are accompanied by large amounts of generated heat (Joule heating). Thus, 20 j. C. Giddings and K. Dahlgren, Sep. Sci. 6, 345 (1971).
466
PURIFICATION PROCEDURES; ELECTROPHORETIC METHODS
[35]
there are limitations on the magnitudes of the electric fields which can be applied. This is partly because resolution decreases with increasing temperature (since diffusion coefficients increase with temperature) and partly because gels can actually get hot enough to burn. Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat than thick ones and are therefore capable of higher resolution. Electric fields used in IEF are generally of the order of 100 V/cm.
Gradient Instability For most practical purposes, pH gradients are stable once the steady state has been reached. However, during extended focusing runs (longer than about 3 hr under standard analytical conditions), gradients are found to slowly deteriorate.l-4,7 This decay is characterized by a drift of the gradients toward the cathode and is accompanied by acidification at the anode, flattening of the gradient in the neutral pH region, and a loss of alkaline bands. The mechanism of the instability, which has been called "cathodic drift," is not completely understood. Cathodic drift is probably caused by a combination of factors including electroendoosmosis, 21 COz absorption, 22 and nonzero, pI-dependent electrophoretic fluxes. 23 The practical consequence of cathodic drift is that excessively long IEF runs should be avoided.
Additives Many protein samples require the use of detergents for their solubilization. For IEF work, the zwitterionic detergents CHAPS and CHAPSO, or the nonionic detergent octylglucoside at concentrations of I-2% in the gel are recommended. Consult Refs. 1-4 and 24 for details. Even in the presence of detergents, some samples may have stringent salt requirements. Only if salt is an absolute requirement should it be present in a sample, and substantial band distortions should be expected. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts. Urea is a common solubilizing agent, especially for those proteins which precipitate at their isoelectric points, even though it denatures proteins.l-4 Urea (3M) is often found satisfactory for maintaining protein 2~ H. Rilbe, in "Electrofocusing and Isotachophoresis" (B. J. Radola and D. Graesslin, eds.), p. 35. de Gruyter, Berlin, 1977. 22 H. Delincre and B. J. Radola, Anal. Biochem. 90, 609 (1978). 23 R. A. Mosher, W. Thormann, and M. Bier, J. Chromatogr. 351, 31 (1986). z4 L. M. Hjelmeland and A. Chrambach, Electrophoresis 2~ 1 (1981).
[35]
ISOELECTRICFOCUSING
467
solubility, but concentrations up to 8 M urea have been used. Only fresh solutions of urea, treated with a mixed bed ion-exchange resin, should be used in order to prevent carbamylation of amine and sulfhydryl groups of proteins. Experimental Procedure The following protocol describes the use of polyacrylamide slabs for IEF on horizontal flat-bed cells. Gel preparation, sample application, focusing conditions, and detection methods are included. The gel recipe is for 12 ml of 5% T (3% C) acrylamide, 2% ampholytes, and 5% glycerol. This is sufficient for casting one standard-size gel of 100 x 125 × 0.8 mm (10 ml) or four 100 × 125 × 0.2 mm gels (10 ml total). The thinner gels can be run at twice the voltage of the thicker ones for increased resolution. Equipment and reagents for IEF are available from many suppliers. For best results, follow the manufacturer's instructions and recommendations, especially when working with high-voltage equipment. Except where noted, reagents for IEF can be prepared as concentrated stock solutions. All water used should be distilled or deionized. Stock Solutions Acrylamide monomer concentrate (25% T, 3% C): Dissolve 24.25 g acrylamide and 0.75 g bisacrylamide in about 70 ml of water. Adjust the final volume to 100 ml. Filter through a 0.45-ttm filter. Store protected from light at 4 ° for up to 1 month. Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, do not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder or solutions containing it. For disposal of unused acrylamide, add bisacrylamide (if none is present), induce polymerization, and discard the solidified gel 25% glycerol (w/v): Weigh 25 g glycerol in a beaker. Add about 50 ml of water and mix well. Dilute to 100 ml with water Carrier ampholytes: Use ampholytes undiluted unless instructed otherwise by the manufacturer. Carrier ampholytes are supplied as aqueous solutions, usually containing 40 or 20% (w/v) solids. The pH range used will depend on the protein(s) of interest 0.1% (w/v) riboflavin 5'-phosphate (FMN): Dissolve 50 mg riboflavin 5'-phosphate in 50 ml water. Store protected from light at 4 ° for up to 1 month 10% (w/v) ammonium persulfate (APS): Dissolve 100 mg APS in 1 ml of water. Prepare this solution fresh daily
468
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
TEMED (N,N,N',N'-tetramethylethylenediamine): Use TEMED undiluted as supplied. Use only pure, distilled TEMED. Store cool and protected from light
Casting Gels The use of gel support film for polyacrylamide is highly recommended, especially with thin gels, which cannot be easily handled unless supported. Polyacrylamide binds covalently to these sheets of treated polyester, 2,25 simplifying gel handling in all steps, from running gels through drying and storing them. Although polyacrylamide gels adhere to wellcleaned glass plates and remain bound through the IEF runs, gels will come off of the backing plates during the staining or destaining steps. IEF gels are very difficult to manipulate once they become detached from their backings. Basic ampholytes (pH > 8) may interfere with the adhesion of gels to support films. Increasing the APS concentration in the final gel to 0.7 mg/ml (84/xl of 10% APS/12 ml of the gel solution given below) should alleviate the problem. Prolonged soaking in the acidic staining and destaining solutions can also affect adhesion of polyacrylamide gels to the support films. Do not soak the gels any longer than necessary in the staining and destaining solutions. 1. Place a few drops of water on a clean glass IEF plate and place the hydrophobic side of a gel support film against the plate (water beads on the hydrophobic sides of the films). Roll the support film fiat with a test tube or similar object to force out excess water and air bubbles and wipe off excess liquid at the edges. Capillarity is sufficient to hold the supported gel on the plate throughout the run. Place the glass plate on the casting tray with the gel support film facing down. 2. Prepare monomer-ampholyte solution from the stock reagents: Water Monomer concentrate 25% (w/v) glycerol 40% ampholyte (w/v)
6.6 2.4 2.4 0.6
ml ml ml ml
Adjust the volume of water to accommodate additives and different ampholyte concentrations; e.g., with 20% ampholytes use 6 ml of water and 1.2 ml of ampholyte solution. 3. Deaerate the monomer-ampholyte solution under vacuum for 5 min; e.g., in a bell jar, desiccator, or vacuum flask. 25 B. J. Radola,
Electrophoresis 1,
43 (1980).
[35]
ISOELECTRICFOCUSING
469
4. Add initiators, swirling gently to mix them into the monomerampholyte solution. 0.1% (w/v) FMN 10% (w/v) APS TEMED (undiluted)
60/zl 18 tzl 4/zl
5. Using a pipet and bulb, carefully introduce the monomer solution between the support film-glass plate and the casting tray. Control the flow to prevent air bubbles. If a bubble becomes trapped in the monomer solution, slide the plate sideways until the bubble escapes at an edge, then reposition the plate so that there is a uniform layer of monomer under it. 6. Position a fluorescent lamp directly over the tray about 3-4 cm from the gel. Illuminate the solution for about 45 min. 7. Lift the gel from the tray by gently prying it up with a spatula. Turn the plate over, with the gel upward, and illuminate the gel for a further 20 min to polymerize monomer remaining on the gel surface. 8. The gel may be used immediately or it can be covered in plastic wrap and stored at 4 ° for several days. Best results are sometimes obtained by letting a gel " c u r e " (polymerize completely) overnight at 4 ° before use.
Sample Preparation Protein samples for IEF must be substantially salt free and free of precipitates. Small samples (1 to 10 tzl) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable sample solutions can be prepared by dialysis or gel filtration. Good visualization of focused lanes generally requires a minimum of 0.5 /xg of protein/band with dye staining or 50 ng of protein/band with silver staining (see below).
Sample Application There are many suitable methods for applying samples to thin-layer polyacrylamide gels. ~-4One of the simplest methods is to place filter paper strips impregnated with sample directly on the gel surface. Up to 25 tzl of sample solution can be conveniently applied after absorption into 1-cm squares of filter paper. A convenient size for applicator papers is 0.2 x 1 cm, holding 5/zl of sample solution. There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH
470
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
the samples should not be applied closer than 1 cm from either electrode. Preforming the pH gradient before sample application will also limit the exposure of proteins to pH extremes. Some polypeptides are eluted most efficiently from strips when applied to the anodic side of the ge1.1,4,26 A good strategy when focusing a protein for the first time is to apply samples to three different areas of the gel, one near each electrode and one near the middle of the gel. This strategy is also valuable in estimating the approach to steady-state focusing. 3,22 When the patterns obtained on applying the sample at opposite ends of the gel become identical, the steady state can be assumed to have been reached. Steady-state conditions should be duplicated when determining the pl values of particular proteins. It should be noted that samples applied at opposite ends of IEF gels will not always focus into identical patterns. The reason for this is not clear, but may be related to interactions of proteins with differing species of carrier ampholytes during focusing.
Focusing 1. Connect theelectrophoresis cell to a refrigerated circulator cooled to 4 °. 2. Wet the cooling stage with a few drops of water or 0.5% glycerol to ensure good thermal contact with the gel backing. Place the gel on the platform (gel upward). Blot any excess liquid from the cooling stage to eliminate possible electrical shorting paths. 3. Cut electrode strips. Thick filter paper or (uncolored) blotter paper work well as electrode strips. More than one thickness can be used. The strips should be about 7 mm wide and cut about 4 mm shorter than the width of the gel (this prevents electrical arcing to the cooling stage). Place the electrode strips on a glass plate and wet them with the appropriate electrolyte solutions. The anode solution is acidic and the cathode solution is basic. a. For most purposes, use 1 N NaOH as catholyte (negative terminal) and 1 N H3PO4 as anolyte (positive terminal).1-4 b. At high voltages, as in focusing with ultrathin gels (0.2 mm or less), the following electrolytes are recommended25: Catholyte: 20 mM lysine, 20 mM arginine, 2 M ethylenediamine. Dissolve 0.36 g lysine (free base), 0.34 g arginine (free base), and 13.4 ml ethylenediamine in water to give 100 ml. Two molar ethanolamine (12 ml/100 ml) can be substituted for the ethylenediamine. Store at 4 °. 26 p. G. Righetti and F. Chillemi, J. Chromatogr. 157, 243 (1978).
[35]
ISOELECTRIC FOCUSING
471
Anolyte: 20 mM aspartic acid, 20 mM glutamic acid. Dissolve 0.26 g aspartic acid and 0.29 g glutamic acid in water to give 100 ml of solution. Store at 4° . 4. Blot the wetted electrode strips with paper towels until they are slightly moist. Carefully place the strips along the appropriate edges of the gel. Do not allow the strips to extend beyond the sides of the gel. 5. Cut 0.2 × 1 cm pieces of filter paper for sample application. Place the pieces of paper on a glass plate and pipette 5/~l of a protein sample solution to each piece. Place the applicator papers on the gel l cm from the anode (sample application is discussed above). 6. Position the electrodes over the electrolyte strips, making sure that there is good contact between the electrodes and the strips. 7. Set the power supply to constant power and run at 6 W/gel, with a voltage limit of 1500 V for standard size gels (100 x 125 x 0.8 mm). Total run time is about 2 hr. The actual running conditions will vary with the apparatus, the gel thickness, the sample solution, and the ampholytes. Gels should always be run at the highest voltage compatible with the heat-dissipation capabilities of the electrophoresis cell. Check the manufacturer's recommendations for proper power settings. At the start of a run, when voltage is first applied, the current will be at its highest value, because the carrier ampholytes have not yet focused. As the run progresses, the conductivity of the gel will drop and the current will fall. It is recommended that the run be started in the constant power mode set at the heat-dissipation limit of the cell. The power supply should be maintained in this mode until the current drops to its lowest value and the highest voltage is reached. (Recall that power = voltage x current and current = voltage x conductivity.) Standard-size gels run at 6 W usually plateau at 1200-1500 V in about l hr. When the maximum voltage is reached, switch to constant voltage regulation (at the voltage limit) for the remainder of the focusing run. The paper application strips can be removed at the time the switch to the constant voltage mode is made. A good way to monitor the progress of a run is with colored marker proteins. However, not all proteins focus at the same rate, so that for accuracy and reproducibility, it is necessary to predetermine the correct conditions for reaching the steady state for the protein of interest. Initial power settings of l0 W/100 × 125 mm gel are common, and final voltage gradients as high as 300 V/cm interelectrode distance have been used with thin gels (0.2 mm). It is customary to characterize the extent of focusing in IEF runs with the time integral of the applied voltage, expressed in volt-hours. 2,22 The volt-hour designation is meant as a standard for reproducing focusing
472
PURIFICATION PROCEDURES; ELECTROPHORETIC METHODS
[35]
conditions. The conditions for attaining steady-state focusing, once determined, are reproducible. However, many factors, especially temperature, affect the absolute reproducibility of focusing. Thus, although the volthour quantity is a convenient indicator of the extent of focusing, it is not a definitive measure of the IEF process. High voltages run for short times result in better separations than low voltages and long times. Detection of Protein Bands Protein staining is the most general method of detection. Discussions of other detection methods and means for quantitating protein bands in gels can be found in Refs. 1-4. Carry out staining and destaining steps at room temperature with gentle agitation (e.g., on an orbital shaker platform) in any convenient container, such as a glass casserole or photography tray. Carefully peel off sample paper strips and electrode wicks (if possible) before beginning the procedure. Standard Procedure. No preliminary fixation step is required with this method. Bands containing proteins in microgram quantities are easily seen. 1. Prepare the staining solution: 0.04% Coomassie Brilliant Blue R-250, 0.05% Crocein Scarlet, 0.5% CuSO4 in 27% ethanol, 10% acetic acid: Water CuSO4 Ethanol Glacial acetic acid Coomassie Brilliant Blue R-250 Crocein Scarlet
630 5.0 270 100 0.4 0.5
ml g ml ml g g
2-Propanol can be substituted for ethanol. Dissolve the cupric sulfate in the water before adding the alcohol. Add the dyes to the solution last. Filter the solution after the dyes have dissolved. The staining solution is reusable. Store it at room temperature. Crocein Scarlet rapidly binds and fixes proteins. 27 Cupric sulfate enhances stain intensity. 1,4,28 2. Soak gels in staining solution for at least 1 hr. 3. Destain with a large excess of 12% ethanol. 7% acetic acid, 0.5% CuSO4 (810 ml H20, 5 g CuSO4, 120 ml ethanol, 70 ml acetic acid) until a clear background is obtained. This will require several changes of destaining solution. Grainy precipitates of dye will sometimes settle on the surfaces of gels after the staining solution has been reused several times. 27 A. J. Crowle and L. J. Cline, J. lmmunol. Methods 17, 379 (1977). 28 p. G. Righetti and J. W. Drysdale, J. Chromatogr. 98, 271 (1974).
[35]
ISOELECTRICFOCUSING
473
These precipitates can be wiped off of the gel with a gloved finger or a moistened tissue while the gel is in the destaining solution. 4. Soak the gel in 12% ethanol, 7% acetic acid to remove the cupric sulfate. Crocein Scarlet can be omitted from the staining solution. If so, gels must be immersed in fixative (4% sulfosalicylic acid, 12.5% trichloroacetic acid, 30% methanol) for at least 30 min prior to staining. If ultra-thin (0.2 mm) gels detach from gel support film during the staining procedure, shorten immersion times, so that the total staining and destaining takes no more than 2 hr. Quick Stain. The following technique 29 is nearly as sensitive as the above one and requires no destaining (it cannot be used in the presence of detergents): Immerse the gel for 1 hr in 3.5% perchloric acid containing 0.025% Coomassie Brilliant Blue G-250. For intensification, immerse the gel in 7% acetic acid. Silver Stain. Silver staining is 10 to 100 times more sensitive than dye staining. Before beginning the silver staining procedure, IEF gels must first be fixed in 30% methanol, 10% trichloroacetic acid, 3.5% sulfosalicylic acid for 1 hr, followed by at least 2 hr in several volumes of 30% methanol, 12% trichloroacetic acid. The Merril silver staining protocol 2,14,30 must be modified for IEF gels bonded to gel support film to include two soaks for 5 min each in 400 ml deionized water between the oxidizer and silver reagent steps, and a 1-min wash in 400 ml of water between the silver reagent and developer steps.
Preservation of Gels To dry gels on support film, simply allow them to dry overnight in air in a dust-free location. Alternatively, gels can be carefully dried with a heat gun at a low setting. To dry unsupported gels, first soak them in 7% acetic acid, 5% glycerol for 1 hr, then smooth them on water-wetted filter paper and dry them in a gel dryer.
Determining pH Gradients When focusing is completed, pH gradients can be determined in various ways. The most straightforward method is to base pH profiles on the 29 A. H. Reisner, P. Nemes, and C. Bucholtz, Anal. Biochem. 64, 509 (1975); see also A. H. Reisner, this series, Vol. 104, p. 439. 30 C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Science 211, 1437 (1981); see also C. R. Merril, D. Goldman, and M. L. Van Keuren, this series, Vol. 104, p. 441; C. R. Merril, this volume [36].
474
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
positions of focused marker proteins. Extensive, but outdated, tables of protein isoelectric p o i n t s 31-33 c a n be consulted in choosing appropriate markers for a particular experiment. It is much simpler, though, to make use of one of the many commercially available protein mixtures. 1EF protein standards are combinations of proteins with well-characterized pl values blended to give uniform staining. The blends often contain naturally colored proteins which allow focusing runs to be continually monitored. They also usually contain proteins that achieve steady-state focusing in relatively short times. Marker proteins usually reach the steady state in about 2500 V-hr. Gels are calibrated with one or two lanes of IEF protein standards. Unknown isoelectric points can be interpolated from graphs of the positions of focused marker proteins plotted as functions of their isoelectric points. pH gradients can also be directly determined with surface electrodes or by elution of ampholytes (before staining). In the latter method, first either slice gels with a blade or punch them into closely spaced pieces with a sharp cork borer. Individually soak each piece of gel in a minimum volume of degassed water or 10 mM KCI for 1-2 hr and measure the pH of each solution. Regardless of the method used in determining the gradient, what is actually measured is the pH of the focused carrier ampholytes, not the proteins themselves. Ideally, pH measurements should be made at the same temperature as the IEF run. Nevertheless, temperature and solvent effects and interference from absorption of atmospheric CO2 are usually neglected in most pH determinations unless accurate pl measurements are required. Discussions of the effects of these factors in Refs. 1, 3, 4, 7, and 22 should be consulted. Microheterogeneity and Artifacts. Multiband IEF patterns can arise from molecular interactions and conformation changes as well as from inherent isoelectric microheterogeneity. 34 Ampholytes can reversibly bind directly to proteins, proteins can undergo sequential pH-dependent conformational changes, and proteins can interact with one another. These types of reactions can artifactually alter the pI profiles o f proteins. On the other hand, many proteins are inherently heterogeneous, consisting of isoelectric isomers. To distinguish between artifactual and inherent heterogeneity, single focused bands should be cut out and rerun. If a 31 p. G. Righetti and T. Caravaggio, J. Chromatogr. 127, 1 (1976). 32 D. Malamud and J. W. Drysdale, Anal. Biochem. 86, 620 (1978). 33 p. G. Righetti and G. Tudor, J. Chromatogr. 220, 115 (1981). J. R. Cann, in "Electrokinetic Separation Methods" (P. G. Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 369. Elsevier, Amsterdam, 1979; see also J. R. Cann, this series, Vol. 61, p. 142.
[35]
ISOELECTRICFOCUSING
475
single band splits into multiple bands on refocusing, artifact formation is indicated. When rerunning a band, care should be taken to rerun it under the same conditions and from the same position on the gel as the initial sample. Preparative Isoelectric Focusing Two techniques which are useful for laboratory-scale preparative electrofocusing are IEF in granular beds I-4,6,11,35 and use of the Rotofor cell. 12,~3,36Both methods allow preparative fractionations on the scale of from hundreds of milligrams to grams of protein, with recoveries of greater than 90% possible. 11 Purification levels between 10- and 100-fold place IEF methods intermediate between ion-exchange and ligand-binding chromatographies as preparative methods. IEF is well suited for use at any stage of a preparative scheme, and is particularly effective in the early stages of purification. In many cases, simple sequential fractionation and refractionation on the same device provides the desired purity. It is not necessary to attain steady-state focusing in preparative IEF, since adequate separations may be achieved before then. IEF in Granulated Gel Beds
Focusing in beds of granulated polyacrylamide or dextran allows highresolution separation and recovery of relatively large quantities of protein. ,-4.6,11,35Granular polyacrylamide gels are recommended for this procedure because of low residual charge and resistance to enzymatic degradation. 11Focusing is carried out in ampholyte-containing gel slurries in specially designed trays in standard fiat-bed apparatus. The manufacturer's instructions should be followed for proper use of each particular cell. The Rotofor Cell
The easiest to use preparative electrophoresis device is the Rotofor cell developed by Egen, Bier, and associates 1z,13(available only from BioRad Laboratories). The principle of the Rotofor cell, IEF in free solution, is similar to that of column methods. However, zone stabilization in the Rotofor cell is achieved not by means of density gradients, but by turning the column on its side. Gravitationally induced convection is inhibited by rotating the column about its (horizontal) axis. The separation column is 35 C. Demeulemester, G. Peltre, D. Panheleux, and B. David, Electrophoresis 7, 518 (1986). 36 N. B. Egen, M. Bliss, M. Mayersohn, S. M. Owens, L. Arnold, and M. Bier, Anal. Biochem. 172, 488 (1988).
476
PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS
[35]
divided into compartments by means of screens of woven polyester. The screens offer resistance to fluid convection, but do not hinder the flow of current or the transport of proteins. Proteins, which are initially dispersed uniformly throughout the chamber, migrate to the one or more compartments which are at pH values nearest to their isoelectric points. The combined effect of compartmentalization and rotation is superior to either method alone in maintaining the stability of focused zones. The segmentation of the column also facilitates fraction collection. A focusing chamber, capable of holding up to 55 ml of sample, is divided into 20 compartments by a core made up of 19 disks of polyester screen (6-/~m pores). A ceramic cooling finger runs through the center of the focusing chamber to dissipate the heat generated during the run. Two electrode assemblies hold the anolyte and catholyte solutions. Appropriate ion-exchange membranes and gaskets isolate the electrolytes from the sample in the focusing chamber while allowing electrical contact with the material in the chamber. The anolyte is usually 0.1 M H3PO4 and the catholyte is usually 0.1 M NaOH, but any other electrolytes which are compatible with IEF can be used. Vent caps provide pressure relief from the gases which build up in the electrode chambers by electrolysis during the run. The entire assembly rotates around the central horizontal axis during the run to inhibit convection, maintain even cooling and efficient electrical contact, and prevent the screens from becoming clogged by precipitated protein. Runs are at 4 ° at constant power (12 W) for 4 hr. Simple and rapid sample collection is by aspiration through tubing lines connecting the 20 individual compartments with corresponding test tubes in a vacuum chamber. Collection is accomplished in seconds, minimizing remixing of fractions by diffusion. Some remixing of adjacent zones takes place, however, because of the finite dimensions of the fraction compartments. The individual test tube fractions are easily sampled for assay or measured for pH with standard electrodes. Samples for the Rotofor need not be completely desalted before fractionation. Ions in the sample solution will be electrophoresed into the two end compartments in the early stages of the run. Carrier ampholyte (2%, w/v) in the initial sample solution supplies enough ampholyte for refractionation of pooled material. After the tub~s containing the protein of interest have been identified, the assay peak can be pooled for a second run. The amount of carrier ampholytes contained in the pooled fractions is adequate for refractionation. The pH range covered on refractionation is determined by the pooled fractions and is generally much narrower than the initial range. Twenty-five-fold purification in a single run and 1000-fold purification by refractionation have been achieved. The ideal sample run on the Rotofor cell would contain only the pro-
[36]
GEL-STAININGTECHNIQUES
477
tein mixture, water, and ampholytes. However, pI precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°. Detergents (1-2%, w/v) may also be added to samples. Zwitterionic detergents such as CHAPS, CHAPSO, and nonionic octylglucoside are satisfactory. Triton X-100 and NP-40 may be less satisfactory due to their slight charge content.
Removal of Ampholytes from Proteins There are a number of ways to separate ampholytes from proteins.l-4 Electrophoresis, ammonium sulfate precipitation, and gel filtration, ionexchange, and hydroxylapatite chromatographies have all been used. Dialysis is a simple and effective method for removing ampholytes from solutions of proteins. First, adjust the pooled fractions to 1 M NaCi to disrupt weak electrostatic complexes between ampholytes and proteins, then dialyze the solutions into appropriate buffers. Extensive dialysis is required for thorough removal of ampholytes. There is no good way to demonstrate complete absence of ampholytes in a protein solution, but for many applications they need not be removed.
[36] G e l - S t a i n i n g T e c h n i q u e s
By CARL R. MERRIL Protein Stains Naturally colored proteins such as myoglobin, hemoglobin, ferritin, and cytochrome ¢ may be directly observed in gels illuminated with light in the visual spectrum, providing that their chromophores are not damaged during electrophoresis. 1However, the visualization of most proteins requires the use of dyes or stains. Organic stains were first utilized for the detection of proteins on gels. Recently metal-based stains, such as the silver stains, have achieved widespread use because of their increased sensitivity. A number of organic stains have been adapted for the detection of electrophoretically separated proteins, including Bromphenol Blue, 2 Fast Green (Food Green 3) and Amido Black (Acid Black 1). 3 Some of these 1 B. D, Davis and E. J. Cohn, Ann. N.Y. Acad. Sci. 39, 209 (1939). 2 E. L, Durrum, J. Am. Chem. Soc. 72, 2943 (1950). W. Grassman and K. Hannig, Z. Physiol. Chem. 290, 1 (1952).
METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
