In the Laboratory
A Convenient and Highly Specific Western Blot Experiment for Introductory Biochemistry
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Christopher J. Fenk* and Stephanie Y. Grooms Chemistry Department, Kent State University–East Liverpool Campus, East Liverpool, OH 43920; *
[email protected] Donald G. Gerbig Jr. Biology Department, Kent State University–East Liverpool Campus, East Liverpool, OH 43920
Rationale Immunoenzymatic staining of membranes, or immunoblotting, is a powerful biochemical technique used to determine the presence of antigens in biological samples (1, 2). This method of immunoassay is widely used in clinical and research laboratories as a diagnostic tool, making its introduction into the undergraduate curriculum extremely important. For example, the Western blot procedure for protein identification is currently used in the detection or confirmation of Lyme disease (3) and HIV infection (4 ). An added benefit of incorporating a Western blot experiment into introductory biochemistry curricula is that numerous chemical and biochemical concepts are efficiently demonstrated. The principal concepts of gel electrophoresis, antibody–antigen binding, genetic engineering, and protein structure are readily introduced by this procedure. In addition, fundamental concepts of electrochemistry, hydrogen bonding and intermolecular attractions may be reinforced. Unfortunately, undergraduate students typically do not gain experience with this important biochemical testing method. This is due to several factors, not the least of which are the relatively high cost of materials and the extensive laboratory time necessary to complete a traditional Western blot immunoassay. Coupling of recent technological advancements with several procedural improvements resulted in the development of a particularly time- and cost-effective protocol. The Western blot experiment described herein is rapid, selective, and extremely sensitive and uses safe, readily available reagents. It allows students to separate a complex mixture of proteins via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using a precast 4–20% gradient gel. The proteins are then transferred to a nonflammable support matrix and a single protein is identified using immunological reagents in a manner analogous to the HIV Western blot confirmatory test. The entire procedure may be conveniently conducted in two consecutive 3-hour laboratory periods using a single 10–12-well polyacrylamide gel for each group of four to six students. Although similar to the commercial HIV Western blot confirmatory test, this experiment has several important differences. This procedure does not involve the use of proteins derived from infectious agents or human sera and is consequently safer for a student investigation. In addition, one
protein band is visualized instead of a minimum of two bands in the HIV test (5). This greatly simplifies student interpretation of experimental results. Experimental Procedure Students begin the experiment with a mixture of 13 proteins. Their principal experimental objectives are to identify an unknown protein in the mixture (bovine serum albumin, BSA) and to determine its relative molecular weight. The protein mixture is denatured, then separated in duplicate by SDS–PAGE. Optimum results are obtained using a precast 4–20% gradient gel. Directly after separation, the proteins are electrophoretically transferred (electroblotted) to a support membrane composed of poly(vinylidine difluoride) (PVDF), a nonflammable alternative to nitrocellulose. The separation and protein transfer can be readily accomplished in three hours. At this point in the experiment, the PVDF membrane may be air-dried and stored in a plastic container until the next laboratory period. The PVDF membrane is then cut into strips, separating the parallel experiments. One set of membrane strips is directly stained with India ink in order to visualize all the transferred proteins. The India-ink-stained membranes are then compared to a reference gel stained with Coomassie blue, to determine retention factors (R f’s) for the major protein bands. These R f values are then used by the students to create a calibration curve that will enable them to determine the molecular weight of the “unknown” protein, bovine serum albumin. The other membrane strips are treated with porcine gelatin to block the remaining binding sites. The transferred proteins are then incubated with a primary antibody selective for bovine serum albumin. The primary antibody, rabbit polyclonal serum (rabbit anti-bovine albumin), is then detected using a secondary binding protein, recombinant engineered protein G horseradish peroxidase conjugate (recombinant engineered protein G-HRP). The secondary binding protein is specific for IgG antibodies such as those found in the rabbit polyclonal serum and is easily visualized by reaction with hydrogen peroxide and either diaminobenzidine (DAB) or tetramethylbenzidine (TMB). • C AUTION : DAB and TMB are derivatives of benzidine, which is a known carcinogen. Use care when handling these reagents.
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In the Laboratory
The R f value of the developed band is compared to the calibration curve prepared by the students, allowing bovine serum albumin to be identified and its molecular weight determined. Results and Discussion Several aspects of this experiment are noteworthy for an introductory biochemistry laboratory exercise and differ significantly from a previously reported Western blot experiment (6 ). A commercially available mixture of proteins, SDS–PAGE standards, is employed for this experiment. This eliminates the need to optimize protein concentrations for gel loading and immunological detection. This experiment also utilizes precast gradient gels, which are extremely convenient and have several distinct advantages. Gradient gels have superior resolving power compared to standard gels. The time requirement, cost, and hazards (7 ) related to the preparation of polyacrylamide gels are also avoided. In addition, precast gels offer highly reproducible experimental results. By not consuming valuable laboratory time for the mechanical pouring of gels, students have more time to investigate the significance of the experimental results. Use of recombinant-engineered protein G-HRP is also an important aspect of this experiment. Traditionally, Western blots employ enzyme-labeled secondary antibodies that bind to the primary antiserum. Unfortunately, secondary antisera may lack antigen specificity, giving rise to background bands. Enzyme-labeled bacterial cell wall proteins such as recombinant engineered protein G minimize nonspecific reactivity to the antigens transferred to the blots and are excellent substitutes for secondary antibody reagents (8). This binding selectivity exemplifies the importance of recombinant DNA technology in modern laboratories and provides students the opportunity to investigate the topic of genetic engineering. Finally, the primary antibody and secondary binding protein titers used in this experiment are extraordinarily dilute (1/32,000 and 1/2,000, respectively). This demonstrates the sensitivity of the Western blot technique while significantly reducing the cost of the experiment. This experiment also has several important advantages over commercially available electrophoresis and Western blot kits. Commercial kits often involve electrophoresis using agarose as the separation matrix and provide only modest protein resolution. Polyacrylamide gels, as used in this exercise, give superior resolution of proteins compared to agarose gels. Consequently, a very precise and accurate molecular weight determination of the active protein (BSA) may be achieved using the protocol reported here. In addition, some Western blot kits are simply simulations, relying on electrophoresis of dyes to mimic antigen separation, and do not actually involve antibody–antigen interactions. Students gain valuable experience with modern bioassay techniques during the course of this experiment and learn the importance of reference standards in quantitative assessments. General concepts of antibody–antigen binding as they pertain to protein structure are exemplified and lead to discussions of immunoglobulin binding specificity. The distinction between continuous epitopes and discontinuous epitopes may also be discussed.
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The described Western blot experiment allows for selective detection of bovine serum albumin within a set of 13 proteins by use of highly specific immunological reagents. The entire protocol may be accomplished in two consecutive 3-hour laboratory periods. Importantly, SDS–PAGE and electrophoretic protein transfer may be accomplished in a single laboratory period. Equipment and Chemicals The reagents used in this procedure are relatively inexpensive and readily available from commercial sources. The average cost of disposable items for this experiment is about $7.50 per student. This estimate assumes four students per gel and includes gels, protein standards, PVDF transfer membranes, antibodies, binding proteins, HRP substrate, and buffer solutions. Electrophoresis chambers, power supplies and electrophoretic transfer devices need be purchased only once and are available in most modern biochemistry laboratories. Alternatively, the electrophoresis chamber and power supply may be constructed from inexpensive materials according to published methods (9). Electrophoretic transfer devices may be constructed according to the method of Kyhse-Andersen (10). Acknowledgments We thank Kent State University–East Liverpool Campus for generous support of this project. We also thank Amy S. Goodhart for helpful editorial suggestions and comments. W
Supplemental Material
Supplemental material for this article is available in this issue of JCE Online. Literature Cited 1. Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. USA 1979, 76, 4350–4354. 2. Burnette, W. N. Anal. Biochem. 1981, 112, 195–203. 3. Ziska, M. H.; Donta, S. T.; Demarest, F. C. Infection 1996, 24, 182–186. 4. Burke, D. S. Clin. Lab. Med. 1989, 9, 369–392. 5. Proffitt, M. R.; Yen-Lieberman, B. Infect. Dis. Clin. N. Am. 1993, 7, 203–219. 6. Farrell, S. O.; Farrell, L. E. J. Chem. Educ. 1995, 72, 740–742. 7. See: Smith, E. A.; Oehme, F. W. Rev. Environ. Health 1991, 9, 215–228 and references therein. Acrylamide is a known neurotoxin and a cancer suspect agent, whereas polyacrylamide is nontoxic. 8. Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor: New York, 1988; pp 613–633. 9. Madeira, V. M. C.; Pires, E. M. V. J. Chem. Educ. 1986, 63, 1109– 1111. Hartman, D. R.; Courtney, W. H. J. Chem. Educ. 1990, 67, 703. Brabson, G. D.; Waugh, D. S. J. Chem. Educ. 1986, 63, 540–542. Meyer, J. J. J. Chem. Educ. 1983, 60, 143. Carter, J. B.; Smith, A. D.; Lea, D. J. J. Biol. Educ. 1983, 17, 5–7. 10. Kyhse-Andersen, J. J. Biochem. Biophys. Methods 1984, 10, 203– 209.
Journal of Chemical Education • Vol. 77 No. 3 March 2000 • JChemEd.chem.wisc.edu
