A Fast and Inexpensive Western Blot Experiment for the Undergraduate Laboratory Shawn 0. farrell' and Lynn E. Farrell Colorado State University, Ft. Collins, CO Western blotting is a n important, modern technique for transferring proteins from a gel onto nitrocellulose or other suitable support and then detecting a protein of interest using antib;dies (1,2).Proteins areseparated using eel electronhoresis. The eels are then nlaced aeainst nitrocellulose, which strongly binds proteins. The proteins can be transferred to the nitrocellulose by passive diffusion or with a type of electrophoresis chamber designed for this purpose. The nitrocellulose membranes are then incubated with a blocking solution that contains nonspecific proteins that will bind to the membrane's free binding sites. Antibodies specific to a protein of interest are then incubated with the membrane, followed by extensive washing to remove unbound antibody. A secondary antibody is then used to bind to the primary antibody. This secondary antibody is covalently attached to a n enzyme that can be used for visualization. We have developed a n experiment and optimized the conditions for the undergraduate laboratory. The experiment can be done quickly using a n electrophoretic blotter or more cheaply using passive transfer. Sera from humans and animals are separated on native acrylamide gels. We run the gels with two identical sets of proteins. Half of the gel is stained with Coomassie Blue to localize the proteins. The other half is blotted to transfer the proteins onto the nitrocellulose. A mouse monoclonal antibody against human serum albumin (HSA) is then used to identify HSAor similar proteins on the blot. A secondary goat antimouse IgG labeled with horseradish peroxidase (HRP) is used for detection. This experiment allows the student to learn procedures of vertical gel electrophoresis, staining and destaining of gels, passive transfer or electroblotting, and treatment of nitrocellulose with blocking solutions and antibodies. They also learn how proteins separate by charge-to-mass ratio and how albumins migrate in various bands on a native gel. The specificity of the antibody for the human protein a s compared to the albumins from other species is also studied.
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Flgure I . Acrylamlde gel separation ot anlmal sera. nnlmal sera were separated on a native 10% acrylamide gel and stained with Coomassie Blue as in the Experimental section.The lanescontained (from left to right) sera from human, monkey, pig, horse, and cow. The last lane held BSA.
Experimental Materials Human, monkey, pig, horse, and bovine sera were purchased from Sigma. Mouse monoclonal antibody to human serum albumin (no. A26721 was purchased from Sigma. Antimouse IgG HRP conjugate antibodies (no. W4021) were purchased from Promega. Nitrocellulose was purchased from Schleicher and Schuell. Gel electrophoresis was carried out using a Fisher mini-vertical gel apparatus and FB 105 power supply. Proteins were electroblotted with a Genie Electrophoretic Blotter (Idea Scientific). All other reagents were analytical-grade. 'Author to whom corresoondenceshould be addressed
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Figure 2. Acrylamide gel after passive transfer to nitrocellulose.Anative acrylamide gel was run with animal sera. The proteins were then transferred passively to nitrocellulose for 48 h, and the gel was stained with Coomassie Blue. Methods Gel Electrophoresis Ten-percent acrylamide gels were prepared with a 5% stacking gel as in ref 3 except that all solutions were SDSfree. Human, monkey, pig, horse, and bovine sera were diluted 1:5 in water and mixed one to one with 2x sample
buffer (20% glycerol, 0.13 M Tris, 30 mg% bromophenol blue, pH 6.8). Samples were loaded on one side of the gel in the order above along with 1mglmL electrophoresis-quality BSA also in 2x sample buffer. A second set was then loaded on the other side of the gel. The gels were run a t 150 V for 1h in electrophoresis buffer (0.025 M Tris, 0.192 M glycine, pH 8.3). The gels were removed and cut in half so that each half had a complete set of samples. One half was placed into staining solution (0.05% Coomassie Blue R-250, 25% methanol, 10% acetic acid) and stained overnight with shaking. The gel was destained to the desired background clarity with 10% acetic acid (4-6 h with two solution changes). The other half was saved for transfer to nitrocellulose.
bumin band. After the passive transfer all the proteins except those with the lowest chargelmass ratio were transferred. Figure 3 shows the developed membrane. Only the human and monkey sera had any color, and several bands were visible. Figures 4-6 show the stained gel before electroblotting, after 1 h, and after 2 h of electroblotting. Transfer was complete a t 2 h. The membranes for this ex-
Passive Dansfer The passive-transfer system was similar to a Southern blot transfer. A sandwich of filter paper, the gel, the nitrocellulose, and more filter paper was carefully prepared. All materials were wetted with transfer buffer (electrophoresis buffer with 20% methanol), and a i r bubbles were pressed out. The bottom piece of filter paper acted a s a wick to draw transfer buffer up through the gel and into the nitrocellulose. Paper towels were placed on top of the sandwich to draw the transfer buffer through the nitrocellulose. The proteins were allowed to transfer for 2 days. After the transfer, the gel was stained with Coomassie Blue and destained as above. Eledroblotting A sandwich was made with filter paper, the gel, and the nitrocellulose, and placed into the Genie Electrophoretic Blotter. The proteins were transferred a t 20 V in transfer buffer for 2 h. The gel was then stained with Coomassie Blue and destained a s above. Blocking Excess Binding Sites The nitrocellulose membranes were incubated for 30 min or overnight in 30 mL 10% milk powder in tris buffered saline with ' h e e n 20 (TBST) (10 mM Tris-HCI, 150 mM NaCI, pH 8, 0.05% Tween 20) to block all excess proteinbinding sites on the membrane. The membranes were stored a t 4 "C until used. Reaction with Antibodies These procedures are taken from ref 4. The blocked membranes were rinsed with TBST, covered with 25 mL of mouse anti-HSA primary antibody (1:1000 dilution in TBST), and incubated a t room temperature for 30 min with shaking. The membranes were rinsed brieflv in TBST and then rinsed three times for 10 min each with 30 mL fresh TBST to remove unbound antibodv. The membranes were then incubated fnr 30 min with 25 mL of coat antimouse IEG HRP coniue:lte secondary a&ibody (1:2500 dilution in TBS?). After incubation, t h e membranes were washed a s above to remove unbound sccoudery :~ntihodg Th(r membranes wen, then incuhated for 15 min in 20 ml. of'color devc:lopment solution ( 1 . 0 mg mL 4-chloro-I-napl~th(,I,0 . 2 ' ~ H20?, 2 0 q methanol in TBS, until bands devclop(d
Figure 3. Results of Western Blot of animal sera. The Western blot from the gel shown in Figure 2 was incubated with mouse antwHSA and then goat antimouse IgG H R P conjugate. The protein bands were visualized by reactton with H202 and 4-chloro-1-naphthol. The left-most lane contains the human serum. The second lane contains the monkey serum. No bands from the other lanes were visible.
Results
Figures 1and 2 show the acrylamide gel before and after passive transfer. From left to right the lanes are human serum, monkey serum, pig serum, horse serum, bovine serum, and BSA. Before the transfer the first four lanes show many bands. The large one corresponds to the main al- Figure 4. Acrylarnide gel separation of animal sera. Volume 72 Number 8 August 1995
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Figure 6. Acrylamide gel after 2 h of electroblotting Fioure 5. Acrvlamide ael after 1 h of elecfroblonino. A native acrvla;;lide gel w& run as Gefore. The proteins were theitransferred wiih a Genie Electroblotter at 20 V fir 1 h. periment are not shown because they are identical to Figure 3. Discussion
This experiment allows the student to learn valuable nrocedures currentlv used in biochemistrv and other bioloplcal sclenres The e x p ~ r i r n nwith : acrylam~degel alone 1s valuable. and the diNtmmct!.; between SDS-PAGE and native gel electrophoresis can be studied. We chose a native svstem because the enitope for the mouse monoclonal antibody is not available after denaturing the protein with SDS (5).This had the additional benefit of demonstrating how proteins can have different forms that are separated bv charaelmass ratio. Even the electrophoretic-quality %A showed two dark bands on a native gel. Both the passive transfer and electroblotting techniques are useful to know. With the electroblotting technique, the student can also see the time needed for transfer and how the hightrr c h a ~ ~mas; t : proteins transfer first. The results a1m ; h m how the alhunnns from human and monkey are slmllar: They hoth reacc with the antihudiri to IIS.4, whereas the albumins from the other species are unreactive. For lab classes with long time blocks, this experiment could be done in one day with the electroblotting apparatus. We usually do the experiment in two blocks. Conven-
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ient stopping points occur after the transfer has begun and after the transfer when the membranes are in blocking solution. In most research labs, a n electroblotting apparatus is available, and few try to do passive transfer from acrylamide. With this system, passive transfer is easy and effective, although it does take time. The experiment is reasonably priced, a s well. To run 25 blots would reauire 0.5 mL of primarv antibody, which costs $145. ~he'secondaryantibody HRP conjugate costs $40 for the same number of blots. The minimum technology needed is the vertical mini-gel electrophoresis and power supplies. Fisher, Hoeffer, and Biorad all make similar units in the $300 range. If you are fortunate enough to have one of the many electroblotting apparatuses, you can speed up the experiment considerably. Hoeffer and Idea Scientific both make models that are easy to use and that bold many gel sandwiches simultaneously. We use this experiment for both our major's course and our junior-level course for other life sciences. I t is easily adaptable to almost any schedule. I t is cheap and demonstrates a n invaluable set of techniques and theories. Literature Cited 1. Towbin. H.:Staehelin,T.;Gordon,J . h Nofl. Acod. Sei. USA 1919.76.4350. 2. Burnetre, W N A n o l . Bmchrm. 1981,212.195. 3. Laammh. U.K Nature 1970.227.680. 4. Romega Corporation: Protoblot Western Blot HRPSystem 1988,l-5. 5. Sigma Chemical Catalogue: lmmunoehemicals 1994.1152.
