PROTEIN BLOTTING AND IMMUNODETECTION
While this technology is just becoming feasible, ithas been demonstrated that the antiidiotypic strategy is a potentially powerful approach for the preparation of monoclonal antibodies to receptors which are difficult to isolate in quantities sufficient to be used as antigens. 26 Single-Chain Antibodies via Genetic Engineering Another method of generating homogeneous antibodies to take advantage of or improve their specificity is the genetic engineering of singlechain antibodies. 2s'29 These recombinant molecules consist of the two antibody variable regions connected by a linear peptide. While this technology is in its infancy, the potential for low-cost, high-volume production of highly specific antibodies is considerable. 3° Acknowledgment We wish to acknowledge the expert clerical help in manuscript preparation given us by Ms. Suzanne Mascola. 28 S. Cabilly, A. D. Riggs, H. Pande, J. E. Shively, W. E. Holmes, M. Rey, L, J. Pery, R. Wetzel, and H. L. Heynehey, Proc. Natl. Acad. Sci. U.S.A. 81, 3273 (1984). 29 M. A. Boss, J. H. Kenten, C. R. Wood, and J. S. Emtage, Nucleic Acids Res. 12, 3791 (1984). 30 A. Klausner, Biotechnology 4, 1041 (1986).
 P r o t e i n B l o t t i n g a n d I m m u n o d e t e c t i o n
By THERESE M. TIMMONS and BONNIE S. DUNBAR Polyacrylamide gel electrophoresis (one- and two-dimensional) has become one of the most widely used techniques for the analysis and characterization of complex protein mixtures. 1-5 These gels can be stained directly and proteins visualized by several different methods. 2'3 However, because access to proteins within the matrix is limited, the information i D. Garfin, this volume . 2 B. S. Dunbar, H. Kimura, and T. M. Timmons, this volume . 3 C. R. Merril, this volume . 4 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C., 1988.
METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
gathered about individual components is usually restricted to their molecular weight and pI.6 Electrophoretic transfer of proteins sepa?ated by PAGE onto the surface of an immobilizing membrane makes them more accessible to various reagents and probes and therefore enables further characterization. In addition to direct staining, these "blots" can be probed with lectins, for specific carbohydrate moieties, and with antibodies to identify and characterize antigenic determinants. One powerful application of such protein blots is the identification and characterization of an immobilized antigen by the use of antibody probes, which can be visualized by radiolabeled or enzyme-conjugated second antibodies. (If antibodies are affixed to the membrane, they can be identified and characterized by probing with selected proteins.) A major limitation to this procedure is that the denatured proteins bound to the membrane may no longer contain the same conformational and structural antigenic determinants present in the native protein. Therefore, only antibodies which recognize determinants consisting of a specific amino acid sequence, carbohydrate structure, etc., will be useful in this technique. 4 Protein Electroblotting A wide variety of transfer methods have been developed. 7-9 However, the conditions for optimal transfer and subsequent binding of a specific protein to a membrane must be determined empirically and may vary for different protein samples. Many parameters affect the efficiency of protein transfer, most of which can be easily manipulated. Some will be discussed below. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. Selection of Transfer Membrane. Several types of transfer membranes are now available. In addition to standard nitrocellulose, which is the most commonly used support, 1° several companies now offer nitrocellulose impregnated with a synthetic support, which improves its durability and flexibility without altering its performance. Polyvinylidene difluoride (PVDF) membrane is marketed by Millipore (Bedford, MA), under the trade name Immobilon.11 Although its protein-binding capacity is slightly 6 L. Anderson, "Two-Dimensional Electrophoresis: Operation of the ISO-DALT System." Large Scale Biol. Press, Washington, D.C., 1988. 7 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). s p. Matsudaira, J. Biol. Chem. 262, 10035 (1987). 9 R. Tovey and B. A. Baldo, Electrophoresis 8, 384 (1987). 10 B. Bers and D. Garfin, BioTechniques 3, 276 (1985). 11 M. G. Pluskal, M. B. Przekop, M. R. Kavorian, C. Vecoli, and D. A. Hicks, BioTechniques 4, 272 (1986).
PROTEIN BLOTTING AND IMMUNODETECTION
lower than nitrocellulose, it is mechanically stronger and is compatible with many organic solvents. This allows direct protein staining with Coomassie Blue, and direct amino acid composition and sequence analysis of transferred proteins, without interfering with its subsequent use for antibody probing. Transfer Equipment. Several types of transfer units are commercially available. The Bio-Rad Transblot unit (Richmond, CA), the Hoefer unit (San Francisco, CA), and the Electroblot apparatus (E.C. Apparatus Corp., St. Petersburg, FL) each require 4-6 liters of buffer per experiment, and are routinely used for the efficient and reproducible transfer of proteins. Two gels can be transferred simultaneously using these units, but larger units (Pierce, Rockford, IL) are available to transfer 10-20 gels at once. The Bio-Rad Mini Protean II system contains a small tank transfer unit for the simultaneous blotting of two minigels in 15-30 min. • An alternative transfer apparatus is the semidry electroblotter (Biometra, Bio-Rad, Hoefer, Millipore, and Sartorius, Emeryville, CA) which needs only enough buffer to saturate the filter paper sheets in the gel sandwich. Transfer is complete in 15-30 min. The graphite l~late electrodes present in the early models often resulted in incomplete, patchy, and irreproducible transfer. The plates also were extremely susceptible to pitting and corrosion. However, the electrode plates of some newer models are made of more durable platinum (anode) and stainless steel (cathode). We have had excellent results using the TE70 SemiPhor semidry electroblotter available from Hoefer Scientific, which contains these newer electrodes. Either of the buffers described below for use in the tank transfer units can be used with this instrument. After transfer is complete, the gel can be stained for residual proteins and the membrane can be processed as described below. The use of a tank apparatus is currently preferable for protein transfer applications in which antigen is limited, quantitation is important, regulation of temperature during blotting is required, and time is not a critical factor. If large numbers of blots are needed rapidly, antigen is freely available, and qualitative results are sufficient, semidry electroblotters may be more appropriate. Transfer Buffer. The choice of buffer composition depends on the types of gel and membrane selected. The procedure of Towbin 7 as modified by Anderson 12 specifies a Tris-glycine pH 8.3 buffer containing SDS. The recirculating, ice-cooled, high ionic strength buffer used helps prevent the gel from swelling in the absence of methanol during transfer, which can cause poor resolution of the proteins on the membrane. However, 10 mM t2 N. L. Anderson, S. L. Nance, T. W. Pearson, and N. G. Anderson, Electrophoresis 3, 135 (1982)•
3-[cyclohexylamino]-l-propanesulfonic acid (pH 9.0 or 11.0) plus 10% methanol is suggested by Matsudaira 8 for transfers from SDS-PAGE minigels to PVDF membrane. Although many variations of electrophoretic transfer of proteins to nitrocellulose have been described, we have found that the procedure that omits alcohol from transfer solutions is generally optimal. Because sodium dodecyl sulfate (SDS) is not rapidly removed from the proteins in the absence of alcohol, the detergent-bound proteins are all initially negatively charged and a more quantitative transfer of proteins is achieved. Furthermore, alcohols or other reagents can alter or modify molecules and may therefore destroy some antigenic determinants. Electroblotting Procedure The method described originally by Towbin 7 as modified by Anderson et al. 12 results in efficient and reproducible protein transfer onto either
nitrocellulose (Bio-Rad) and PVDF (Millipore). Electrode buffer: 0.250 M Trizma base 0.192 M glycine Final volume 1 liter
30 g 140 g
Prepare as much buffer as needed to fill the chamber of the tank blotter. Note: This buffer is l0 times more concentrated than most methods suggest, but we have found this results in optimal transfer of most proteins and is required for others. Carry out S D S - P A G E separation of proteins (one- or two-dimensional separations, full-size or minigels). It is usually beneficial to include prestained molecular weight markers: their separation during electrophoresis, and the efficiency of their electrophoretic transfer onto a membrane, can be monitored visually. A variety of these standards are now commercially available. Bethesda Research Laboratories (Gaithersburg, MD) offers blue-stained markers, and Amersham (Arlington Heights, IL) offers "rainb o w " standards (each marker protein can be identified by its own characteristic color dye). It is important to realize that the "rainbow" dyes detach from their respective proteins if they are allowed to remain in solubilization buffer for any length of time. Therefore the standards must be solubilized in a boiling water bath for no more than 60 sec, and i m m e d i ately loaded onto the gel and electrophoresed. Transfer membrane, four sheets of filter paper, and two foam pads are cut to the same size as the gel and soaked in electrode buffer. (If the hydrophobic PVDF membrane is used, it must first be rinsed for a few seconds in 100% methanol and then in water before it is placed in electrode
PROTEIN BLOTTING AND IMMUNODETECTION
buffer. This methanol wetting procedure must be repeated if the membrane is allowed to dry at any point in the transfer and detection process.) The transfer stack is built in the following order: cathode side of unit, foam pad, two sheets of filter paper, gel, membrane, two sheets of filter paper, foam pad, anode side of unit. It is critical to add enough filter paper and foam pads to ensure tight contact of the gel and the membrane in the sandwich unit (for a complete photographic illustration of this procedure, refer to Ref. 4). Close the unit and lower it into the transfer chamber filled with chilled buffer and connect the power supply. Transfer at - 1.2 A for 2½ to 4 hr. (In general, higher acrylamide concentration gels and higher molecular weight proteins will need longer transfer times.) If the buffer warms during the procedure, a recirculating cooling bath may be needed. After transfer is complete, place the membrane into a tray which is slightly larger than the sheet itself to ensure efficient mixing of solutions over the paper. Be sure to place the side of the paper that was next to the gel facing up. The use of prestained markers will help to determine on which side the proteins are immobilized. The transfer is processed as described below. The gel can be fixed and stained to monitor the efficiency of transfer. 3,4 As an alternative, the method described by Matsudaira 8 is effective for transfer of proteins from gels onto PVDF membrane, and is less expensive to use. The equipment and procedure described above is used, but the electrode buffer is 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid (pH 11.0) plus I0% methanol. Transfer is accomplished at 0.5 A for 1030 min. The membrane is processed as described below. Irnmunodeteetion of Proteins The procedure for immunodetection of antigens with antibodies is compatible with either PVDF or nitrocellulose membranes.
Supplies and Reagents Tris-buffered saline (TBS)/azide: 10 mM Tris-HCl, pH 7.0 0.9% NaC1 0.02% Sodium azide Primary antibody, second antibody intermediate (if needed) 125I-Labeled Staphylococcus aureus protein A or protein G (Amersham, ICN, Costa Mesa, CA, NEN, Boston, MA, etc.); 125I-labeled IgG directed against species from which primary antibody is obtained Blocking solution: TBS/azide + 3-5% instant nonfat dry milk (or 3% bovine serum albumin)
Although bovine serum albumin is a more fully characterized and purified reagent than nonfat dry milk, it is expensive to use routinely. A 3-5% solution of nonfat dry milk efficiently blocks most nonspecific binding sites for immunoglobulins. 9'1°'13 However, the carbohydrates present may interfere with binding of an antibody recognizing a carbohydrate determinant. Other common blocking reagents include nonionic detergents such as PVP-40 (polyvinylpyrrolidone, average Mr = 40,000) and Tween 20.10,13,14 Procedure for Immunoblotting. Immediately after protein transfer is completed, place the membrane (protein side up) in a dish and incubate with 100-150 ml blocking solution and shake vigorously at room temperature for 6-24 hr on a rotating or shaking platform that is reliable and can accommodate large numbers of gels. (Note: The best results are achieved when optimal shaking platforms are used. We recommend those available from Pierce Apparatus Branch.) Wash two times with 100-150 ml TBS/ azide for 20 min each. Dissolve the primary antibody in blocking solution, in a volume that will just completely cover the membrane. The amount of antibody will depend on the antibody titer and can range from 20 ~1 to 10 ml of serum in 60 ml of blocking solution. Add the antibody solution and incubate with vigorous shaking for about 6 hr. (Note: These incubation times may be reduced, depending on the titer and nature of the antibodies. However, to obtain the best initial results, we recommend these conditions for optimal signal with low background.) Wash twice as before, and then wash overnight with vigorous shaking. If a second antibody bridge is required (see below), dissolve it in blocking solution and incubate with vigorous shaking for 6 hr. Wash twice as before, then wash overnight and continue with the protein 1251labeling. If no bridge is needed, add lZSI-labeled second antibody or 125I-labeled protein A or G (approximately 10 6 cpm/transfer) in blocking solution and incubate with shaking at room temperature for 6 hr. Protein A and protein G are cell wall proteins isolated from specific bacterial strains, and have specific binding sites for certain classes of immunoglobulins. Protein A binds (to varying degrees) most subclasses of IgG, plus IgM, IgA, and IgD. 15 Protein G binds nearly all subclasses of IgG, but not other classes of immunoglobulins. One important property of intact protein G, as isolated from streptococci group G, is the presence of a separate binding site for albumin, which could give ambiguous results 13 D. A. Johnson, J. W. Gautxch, J. R. Sportsman, and J. H. Elder, Gene Anal. Technol. 1, 3 (1984). ~4 H. Towbin and J. Gordon, J. Immunol. Methods 72, 313 (1984). 15 j. j. Langone, Adv. Immunol. 32, 157 (1982).
PROTEIN BLOTTING AND IMMUNODETECTION
TABLE I PROTEIN A AND RECOMBINANT PROTEIN G RECOGNITION OF IgG FROM VARIOUS ANIMAL SPEC1ES USING DOT IMMUNOBLOT ASSAYS
Recognition by a
Recombinant protein G
+/++ +++ ++ ++ +++ ++ +/+ +/+/-
+ /++ ++ ++ ++ +/++ ++ ++ ++ +/-
species b Mouse Rabbit
Guinea pig Pig Human Cat Dog Goat Cow
Sheep Horse Rat Chicken
+ + + , + + , +, Strong to weak recognition; + / - , very weak; - , no recognition. b Not all subclasses of IgG are recognized by protein A or recombinant protein G.
in immunodetection experiments using tissue homogenates o r s e r u m . 16A7 Recombinant protein G (ICN, Zymed, San Francisco, CA, Bio-Rad, etc.) has been engineered to eliminate this binding site. The species specificity of protein A and recombinant protein G recognition of IgG is summarized in Table I. ~5-1sIf the 125I-labeled protein available does not bind to the IgG of interest, a second antibody bridge can be used: i.e., primary antibody:cat IgG, second antibody:goat anticat IgG, 125I-labeled recombinant protein G. In the case of monoclonal antibodies raised in mouse, we routinely use a rabbit anti-mouse immunoglobulin second antibody bridge, rather than using ~25I-labeled protein A or G directly, to produce the cleanest and strongest signal by autoradiography. The transfer membrane can be air dried before processing by autoradiography or it can be exposed to film while damp. To process the wet 16 j. Bjorck and G. Kronvall, J. Immunol. 133, 969 (1984). 17 S. R. Fahnestock, P. Alexander, J. Nagle, and D. Tilpula, J. Bacteriol. 167, 870 0986). is B. Akerstrom, E. Nielsen, and L. Bjorck, J. Biol. Chem. 262, 13388 (1987).
membrane, drain it and place it on a piece of Whatman filter paper slightly larger than the membrane itself, and wrap in plastic wrap or seal in a plastic bag. After the autoradiogram has been developed, the membrane can be reprobed by exposing it to another primary antibody. If desired, the first primary antibody can be stripped (leaving the immobilized proteins still bound to the membrane) by washing with a low-pH (approximately 2.5) buffer, 19'2°a buffered solution of 0.5% Tween 20 or other detergent, 2° or a high concentration of chaotropic agent such as 3 M NH4SCN. 2° The efficiency of the stripping can be monitored by reexposing the membrane to X-ray film. (This procedure is more effective when the membranes are kept moist during autoradiography.) If the stripped antibodies are neutralized quickly and/or dialyzed exhaustively against TBS, they may be used to probe a second immunoblot. Variations of this technique have been successfully employed to select a specific population of antibodies from a polyclonal antiserum, on the basis of their recognition of a specific antigenic determinant. 19 As an alternative to 1251, antigens can be visualized directly on the transfer membrane using an enzyme-conjugated second antibody, directed against the IgG of the species from which the primary antibody is obtained. (Protein A and protein G are also available conjugated to the enzymes described below.) The enzymes most commonly used in this procedure, alkaline phosphatase and horseradish peroxidase, are coupled to the formation of a colored product which can be detected by visual inspection of the membrane. The high sensitivity of this type of reagent has both advantages and disadvantages. Results are obtained quickly, but the use of an extremely sensitive detection method can be confusing, especially if the background staining level is high. If the signal-to-noise ratio is too low or the optimal amount of protein is not immobilized on the membrane, and the desired information cannot be obtained, the membrane can not easily be reprobed or stripped. However, if 125I-labeled protein A or G is used, the time of autoradiographic exposure can be varied to obtain the optimal signal. The membrane can be reprobed easily, and with less buildup of background signal than is possible with enzyme-conjugated detection. However, the speed of detection is often an overriding concern, and for a familiar antigen-antibody system, the enzyme-conjugated protocol may be the method of choice. The following procedure can be followed for immunodetection by horseradish peroxidase-conjugated second antibodies (DAKO, Santa Barbara, CA, Miles, Naperville, IL, etc.): (1) Block membrane, wash, and incubate in primary antibody as described above; (2) wash twice quickly t9 j. B. Olmsted, J. Biol. Chem. 256, 11955 (1981). 2o D. E. Smith and P. A. Fisher, J. CellBiol. 99, 20 (1984).
PROTEIN BLOTTING AND IMMUNODETECTION
and then overnight in TBS without azide (azide may interfere with the enzyme activity used for antibody detection); (3) dissolve the appropriate peroxidase-conjugated second antibody in blocking solution without azide, and incubate with vigorous shaking for 1 hr at room temperature. The amount of antibody needed will vary with the titer of the preparation used, and must be determined experimentally; (4) wash the transfer membrane for 30 rain in TBS/no azide, with three changes of solution, at room temperature with vigorous shaking; (5) prepare fresh color substrate [20 ml of 100 mM Tris-HC1, pH 7.0, plus 1.0 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (Sigma); 20 ml of 0.02% H202 in H20; 200/~1 of 8.0% NiC12 in H20] (6) mix together and pour over transfer; (7) shake at room temperature until color appears; (8) wash with TBS/azide, and air dry. If immobilized proteins are being used to screen a series of monoclonal antibodies or to characterize antisera or antibodies in limited supply, the miniblotter system (Immunetics, Cambridge, MA) is useful. Antigens are transferred from one-dimensional standard-sized or minigels. The membrane is blocked, and then is clamped in the Lucite holder. The upper surface has open channels that span the height of the membrane, and require as little as 50/xl of primary antibody solution each. Detection of bound antibodies is accomplished by any of the methods described above. Immunoblotting Artifacts Many of the problems encountered with high backgrounds and other artifactual stains on immunoblots can be eliminated. (1) Usually a high background is the result of inadequate blocking of binding sites on the membrane, or inadequate washing. It may be necessary to increase the protein concentration of blocking solution or increase time of incubation with blocking solutions. Be sure that there are sufficient volumes of solutions to cover gels. Even if more dilute solutions are used, better results will be obtained if adequate shaking is used. If an excessive amount of second antibody or labeled probe is used, a high background will frequently be obtained. The system should be optimized to give maximum detection of antigen (signal) without giving high background (noise) (i.e., high signal-to-noise ratio). (2) Uneven background may be due to inadequate washing and can be improved as described above. This can also be a problem if soft plastic dishes are used instead of glass dishes for multiple incubations, or if the membrane is handled improperly. Always use gloves when touching membranes. (3) If little or no antibody binds to the proteins, the antibody may not recognize the denatured form of the antigen, or the antibody titer may be too low. The titer can be increased by affinity purification or concentration of the antibody. (4) Irregular transfer of protein to membranes can be a problem with many tank transfer systems. It is sometimes possible to improve this by adding additional paladium
wire to the chamber used for electrophoretic transfer. Another common problem is that the paper is not pressed tightly enough against the gel. This can be solved by adding additional sponges to compress the gel and paper together. The techniques of polyacrylamide gel electrophoresis, protein electroblotting, and immunodetection combine to provide an extremely powerful and sensitive method for the analysis and characterization of complex protein mixtures. Acknowledgment The authors wish to thank Ms. SuzanneMascolafor expert secretarialassistance.
 I m m u n o p r e c i p i t a t i o n o f P r o t e i n s By GARY L. FIRESTONE and SANDRA D. WINGt;TH
The discovery and use of fixed Staphylococcus aureus (Staph A) as an immunoadsorbent ~-3 has been a major advance in routinely using antibodies as sensitive probes for selectively examining the expression of specific protein products from radiolabeled tissue. The Kessler procedure (and its modified versions) exploits the high adsorption capacity of protein A molecules which are found on the cell walls of certain staphylococci strains, for the Fc region of specific IgG and IgM isotypes. The overall strategy of this procedure involves reacting a small amount of radiolabeled antigen with an excess of antibody followed by the addition of enough fixed Staph A containing protein A to bind all appropriate antibodies regardless of whether they contain bound antigens. The advantage of this procedure (or any protocol that employs antibodies affixed to a solid state matrix) is that an immunoprecipitate per se need not be formed to separate immunocomplexes from cellular polypeptides not recognized by the antibodies. Thus, small absolute amounts of radiolabeled antigens can be rapidly and selectively immunoadsorbed to Staph A pellets and quantitatively fractionated away from the bulk polypeptides by simple low-speed centrifugation. Moreover, the Staph A immunoadsorption method is versatile in that it has proved useful for analysis of soluble as well as membraneassociated polypeptides, since the immunoadsorption is particularly efficient in the presence of either nonionic detergents such as Triton X-100 1 S. W. K e s s l e r , J. Immunol. 115, 1617 (1975). 2 S. W. K e s s l e r , J. Immunol. 117, 1482 (1976). 3 R. D. Ivarie and P. P. Jones, Anal. Biochem. 97, 24 (1979).
METHODS IN ENZYMOLOGY, VOL. 182
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