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The erasable Western blot
ANALYTICAL
BIOCHEMISTRY
161, 89-95
(1987)
The Erasable Western Blot SCOTTH.KAUFMANN,*CHARLES *Oncology
M. EWING,* ANDJOEL. H. SHAPER*.?
Center, Johns Hopkins Hospital and tDepartment Johns Hopkins University School of Medicine,
of Pharmacology and Molecular Baltimore, Mar.vland 21205
Sciences.
Received August 8, 1986 A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a lo-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70°C in 2% (w/v) sodium dodecyl sulfate containing 100 mM &mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed. o 1987 Academic press, IX. KEY WORDS: gel electrophoresis, protein: blotting: immunological methods, nuclear envelope polypeptides; antibodies.
Current methods of immobilizing electro- blots in order to reutilize the antibodies have phoretically separated polypeptides on deriv- been described (e.g., (3-5)), there is no atized paper (reviewed in (1,2)) permit the widely accepted method for removing antirapid analysis of multiple samples for the bodies from blots for the purpose of reusing presence of antigens of interest. There are the blots. In the present study we describe a several situations in which it would be conve- method for removing monoclonal and polynient to sequentially probe a single Western clonal antibodies from dried nitrocellulose blot with different antibodies: (i) when the blots while preserving the immobilized polyimmobilized polypeptides are derived from a peptides. A modification of this method also biological fluid or subcellular fraction which permits reuse of Western blots when proteins is available in small quantities and/or at are immobilized on nylon. great expense, (ii) when a purification scheme is to be monitored simultaneously MATERIALS AND METHODS for the presence of more than one antigen, (iii) when a large number of antibody soluMaterials. Sodium dodecyl sulfate, dithiotions (e.g., hybridoma supernatants or se- threitol, P-mercaptoethanol, and all chemiquential bleeds from a group of subjects) cals for electrophoresis were from Bio-Rad have to be assayed for the presence of anti- (Richmond, CA). Sartorius brand nitrocellubodies against a single antigen or class of an- lose paper (0.45~rcrn pore size) was from tigens, and (iv) when a blot gives an unex- Vangard (Neptune, NJ). Derivatized nylon pected result and probing with a second an- (GeneScreen) was from New England Nutibody will shed light on this result. clear (Boston, MA). Thiodiglycol was from While methods for eluting antibodies from Pierce (Rockford, IL). Bovine serum albu89
0003-2697187
$3.00
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
90
KAUFMANN.
EWING,
min (RIA grade), bovine hemoglobin, and swine skin gelatin were from Sigma (St. Louis, MO). Goat anti-human IgG was from Tago (Burlingame, CA). Goat anti-rabbit IgG, rabbit anti-chicken IgG, and rabbit anti-mouse IgG were from Cappell (Cochranville, PA). Suflers. TS buffer: 10 mM Tris-HCl (pH 7.4 at 20°C) containing 150 mM NaCl. TSM buffer: TS buffer containing 5% (w/v) powdered nonfat milk, 1 mM sodium azide, 100 U/ml penicillin G, and 100 pg/ml streptomycin. Antibodies. Rat liver nuclear envelopes were prepared as previously described (6). Individual polypeptides separated by two-dimensional IEF’/SDS-PAGE (6) were used to immunize chickens according to a schedule previously described (7). The resulting antibodies will be characterized more completely in a subsequent paper. An IgG2, mouse monoclonal antibody against the 94-kDa steroid binding moiety of the rat liver glucocorticoid receptor (antibody No. 7 in Ref. (8)) was kindly provided by Drs. Sam Okret, Ann-Carlotte Wikstriim, and J-A. Gustafsson (Karolinska Institute). An IgG1 mouse monoclonal antibody against poly(ADP-ribose) polymerase (9) was kindly provided by Dr. Guy Poitier (University of Quebec). Rabbit polyclonal antiserum against bovine thymus topoisomerase I and human autoantibody against nuclear envelope polypeptides were kindly provided by Drs. Leroy Liu and Thomas Provost, respectively (Johns Hopkins University). Electrophoresis and transfer. Rat liver nuclei or nuclear envelopes were solubilized, reduced, and alkylated as previously described (6). Samples containing 2.5 X lo6 nuclei or 5 X lo6 nuclear envelopes were separated in the presense of SDS in 0.75-mmthick polyacrylamide gels containing 8% (w/v) acrylamide. Samples were electropho’ Abbreviations sodium dodecyl trophoresis.
used: IEF, isoelectric focusing; SDS, sulfate; PAGE, polyacrylamide gel elec-
AND
SHAPER
retically transferred to nitrocellulose at 4°C in a Hoefer series TE transfer apparatus operating at 90 V for 6- 12 h. Transfer buffer contained 20% (v/v) methanol, 192 mM glytine, 25 mM Tris, and 0.01% (w/v) SDS. All further steps were carried out at room temperature. Polypeptides transferred to nitrocellulose were visualized by staining with fast green FCF (CL 42053) and reequilibrated with TS buffer as previously described (7). The remaining protein binding sites were saturated during a 6- to 8-h incubation in TSM buffer. Incubation with antibodies prior to erasure. All steps were performed at room temperature. The sheets were incubated for 12- 14 h with antibodies diluted in TSM buffer. They were washed three times (15 min each) with TS buffer containing 2 M freshly deionized urea and 0.05% (w/v) Nonidet-P40 and then once (5 min) with TS buffer. Urea was omitted when monoclonal antibodies were used. The sheets were incubated for 60 min with TSM buffer containing 5-10 &i of lz51-labeled secondary antibody (labeled to a specific activity of 5-10 &i/pg by the method of Fraker and Speck (10)) and subjected to sequential 15-min washes (generally three or four) in TS buffer containing 0.05% (w/v) Nonidet-P40 until no radioactivity was detected in the wash buffer. Prior to drying, blots were incubated for 10 min in TSM buffer. They were then dried on absorbent paper at room temperature for 5 min and moved to fresh paper to complete the drying process. (Moving the coated blots after 5 min prevents their sticking to the paper.) They were exposed to Kodak XOmat XAR-5 or XRP-5 film at -70°C as indicated in the figure legends. Erasure of blots. Blots were incubated for 30 min at 70°C with erasure buffer consisting of 2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8 at 20”(Z), and 100 mM @-mercaptoethanol. All further steps were performed at room temperature. After two lo-min washes with TS buffer, the blots were dried (Figs. 1
THE
ERASABLE
and 2) or incubated with TSM buffer for 6 h prior to exposure to a new primary antibody (Fig. 3). Modijcation for use with nylon. With slight modification, the method described above can be used to remove primary and secondary antibodies from Western blots when proteins are immobilized on nylon rather than nitrocellulose. After SDS-PAGE, electrophoretic transfer was performed as described above. Staining with fast green FCF was omitted. The remaining protein binding sites were blocked by a 6- to S-h incubation at room temperature with 10% (w/v) powdered nonfat milk in TS buffer containing 1 mM sodium azide, 100 U/ml penicillin G, and 100 pg/ml streptomycin. After incubation with primary and secondary antibodies, blots were treated with TSM buffer for 10 min prior to drying and autoradiography. Primary and secondary antibodies were removed by a 30-min incubation at 70°C in erasure buffer. After two lo-min washes with TS buffer, blots were recoated for 6-8 h with 10% (w/v) powdered milk in the buffer described above and then exposed to a new primary antibody. RESULTS
D$kdty Erasing Dried Blots A number of investigators have described the elution of antibodies from protein blots using glycine at acid pH (e.g., (3,4)) or 3 M sodium thiocyanate at alkaline pH (5). In these procedures, blots were kept moist during all elution steps to preserve reactivity of the recovered antibodies. We were interested in the possibility of removing antibodies from nitrocellulose blots for the purpose of reusing the blots (e.g., (11,12). In this case, blots with their adherent antibodies were dried at ambient temperature and subjected to autoradiography prior to any attempted removal of the antibodies. When nitrocellulose blots were dried immediately after reaction with primary and secondary antibodies, subsequent treatment for 30 min at 70°C with 2% SDS containing
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100 mM @-mercaptoethanol, with 2% SDS containing 4 M urea, with 3 M sodium thiocyanate, with 0.2 M glycine-HCl (pH 2.5) or with 0.1 M sodium carbonate failed to remove all of the antibody (Fig. 1A, lanes 2-6, respectively). Likewise, treatment with 8 M urea containing 100 mM P-mercaptoethanol and 5 mg/ml bovine serum albumin at 60°C for 60 min (11) failed to remove all of the antibody (Fig. 1A, lane 7). Preliminary experiments also indicated difficulty in removing antibodies when blots had been coated with 3% bovine serum albumin rather than 5% milk prior to incubation with the primary antibody (data not shown). A fraction of the antibody remained tightly adherent to the nitrocellulose even after exposure to strongly denaturing conditions.
Coating with Protein prior to Drying Facilitates Erasure of Nitrocellulose Blots In an attempt to facilitate the subsequent removal of antibody, blots treated with primary and secondary antibody were recoated with 5% (w/v) powdered nonfat milk for 10 min prior to drying and autoradiography. This treatment did not detectably alter the amount of primary and secondary antibody bound to the blot (cf. lane 1, Figs. 1A and 1B). Subsequent treatment for 30 min at 70°C with 2% SDS containing 4 M urea, with 3 M sodium thiocyanate, with 0.2 M glycine (pH 2.5) or with 0.1 M sodium carbonate removed much more of the antibody than previously but still left a weakly detectable signal on the blot (Fig. 1B, lanes 4-7, respectively). Likewise, treatment with 8 M urea containing 100 mM B-mercaptoethanol and 5 mg/ml bovine serum albumin for 60 min at 60°C (11) left a weakly detectable signal (Fig. lB, lane 8). In contrast, treatment with 2% SDS containing 100 mM P-mercaptoethanol removed all of the detectable antibody (Fig. 1B, lane 2). To explore further the mechanism by which milk facilitated subsequent erasure of blots, replicate blots treated with primary and secondary antibody were coated with the
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KAUFMANN,
EWING, AND SHAPER
quently treated with 2% SDS- 100 mM /3mercaptoethanol at 70°C for 30 min to remove antibodies (lane 2 in Figs. 2A-2G). Treatment with dithiothreitol (Fig. 2B) or * thiodiglycol (Fig. 2C) prior to drying did not 31 facilitate the subsequent removal of antibodies from blots. In contrast, treatment with commonly used concentrations of gelatin, B bovine serum albumin, or hemoglobin prior FIG. 1. Coating ofblots with milk prior to drying facihto drying improved subsequent removal of tates subsequent removal of antibodies. Replicate sam- antibody (Fig. 2, panels D-F, respectively). ples of rat liver nuclear envelopes were separated by SDS-PAGE and stained with Coomassie brilliant blue Nevertheless, these treatments left a weakly (lane 0) or transferred to nitrocellulose (panels A and B). detectable signal on the blots. Of the protein solutions tested, milk was most effective at After blocking of unoccupied binding sites in TSM buffer, a single piece of nitrocellulose was treated se- facilitating the subsequent removal of antiquentially with a I:5000 dilution of chicken serum con- body (Fig. 2G). 1234567
123456769
taining antibodies directed against lamin B (upper arrowhead) followed by ‘251-labeled rabbit anti-chicken IgG. After appropriate washes (see Materials and Methods), pieces of the blot were incubated for IO min with TS buffer (panel A) or TSM buffer (panel B) immediately prior to drying. Autoradiography (not shown) confirmed the uniform labeling of lamin B in all lanes. Lanes were then excised and exposed for 30 min at 70°C to 62.5 mM Tris-HCI (pH 6.8 to 20°C) containing 2% (w/v) SDS and 100 mM @-mercaptoethanol (A2, B2, B9), 2% (w/v) SDS without reducing agent (B3), 2% (w/v) SDS and 4 M deionized urea (A3, B4), or 3 M sodium thiocyanate (A4, B5); to 0.2 M glycine-HC1 at pH 2.5 (AS, B6); to 0.1 M sodium carbonate (A6, B7): or for 60 min at 60°C to 8 M deionized urea containing 100 mM &mercaptoethanol and 5 mg/ml bovine serum albumin (A7, B8). After erasure, lane B9 was recoated with TSM buffer and treated sequentially with chicken serum containing antibodies directed against a 47-kDa nuclear envelope polypeptide (lower arrowhead) followed by ‘251labeled rabbit anti-chicken IgG. Lane 1 in each panel served as control. Autoradiography was for 16 days at -70°C with XRP-5 film. Nonadjacent lanes of a single autoradiograph have been juxtaposed to compose this figure. Note that recoating with milk prior to drying did not detectably alter the amount of antibody bound to the blot (cf. A 1 and Bl) but facilitated subsequent removal of antibody (panel B). In panel B all of the treatments except 2% SDS containing 100 mM P-mercaptoethanol (B2, B9) left a faintly detectable signal on the original autoradiograph.
reducing agent dithiothreitol, the antioxidant thiodiglycol, or with a number of different protein solutions prior to drying. None of these treatments detectably altered the amount of antibody bound to the blot (cf. lane 1 in Figs. 2A-2G). Blots were subse-
Repetitive
Use of a Single Blot
To assess the suitability of erased blots for subsequent detection of antigens, a lane
A
BCDEFG
FIG. 2. Effect of various coating solutions on the subsequent removal of antibodies. Replicate samples of rat liver nuclear envelopes on a single sheet of nitrocellulose were treated sequentially with a 1:5000 dilution of chicken serum containing antibodies to lamin B followed by ‘251-labeled anti-chicken IgG. Immediately prior to drying, pieces of nitrocellulose were treated for 10 min with TS buffer (panel A) or TS buffer containing 1 mM dithiothreitol (panel B), 1% (w/v) thiodiglycol (panel C), 0.1% (w/v) gelatin (panel D), 3% (w/v) bovine serum albumin (panel E), 1% (w/v) hemoglobin (panel F), or 5% (w/v) milk (panel G). Autoradiography (not shown) confirmed the uniform labeling of lamin B in all lanes. In each panel, lane 2 was incubated at 70°C with erasure buffer as described under Materials and Methods. Lane 1 served as a control. Autoradiography was for I6 days at -70°C with XRP-5 film. Nonadjacent panels from a single autoradiograph have been juxtaposed to compose this figure. Recoating with any protein solution facilitates subsequent removal of antibodies (panels D-G) but the removal is most complete when blots are treated with 5% (w/v) milk prior to drying (panel G).
THE ERASABLE
erased in 2% SDS containing 100 mM j3mercaptoethanol was recoated with milk and then reacted sequentially with another primary antibody (raised in the same species as the original antibody) followed by radiolabeled secondary antibody (Fig. lB, lane 9). The second antigen was readily detected. In addition, the lack of binding of radiolabel in the region where the original primary antibody was bound confirmed that treatment with 2% SDS containing 100 mM @-mercaptoethanol removed primary antibody as well as radiolabeled secondary antibody. To explore further, the repetitive use of a single blot, polypeptides from the livers of adrenalectomized and glucocorticoid-treated rats were separated by SDS-PAGE, transferred to nitrocellulose, and then reacted sequentially with the following antibodies and their corresponding radiolabeled secondary antibodies: mouse monoclonal antibody to the 94-kDa polypeptide moiety of the rat liver glucocorticoid receptor (Fig. 3A), mouse monoclonal antibody to poly(ADP-
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ribose) polymerase (Fig. 3B), rabbit polyclonal antibody to topoisomerase I (Fig. 3C), human polyclonal antibody to nuclear envelope polypeptides (Fig. 3D), and then fresh aliquots of the two mouse monoclonal antibodies (Figs. 3E and 3F). The results of this experiment are shown in Fig. 3. Consistent with current models of steroid hormone action, the glucocorticoid receptor was detectable in isolated nuclei from hormone-treated rats (Fig. 3A, right lane) but not adrenalectomized rats (Fig. 3A, left lane). The observation that other antibodies bind almost equally to both lanes (Figs. 3B-3D) indicates that the lack of binding of antiglucocorticoid receptor antibody to the sample in the left lane is due to selective absence of this particular antigen rather than aberrant loading or transfer of the polypeptides, thus illustrating one of the potential uses of this method. In addition, these results indicate that the erasing procedure described above permitted removal of various polyclonal antibodies as well as the two monoclonal antibodies.
.
FIG. 3. Repetitive use of a single piece of nitrocellulose. Nuclei from the livers of adrenalectamized (left lane) or glucocorticoid-treated (right lane) rats were subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose. After coating with TSM buffer, a single piece of nitrocellulose was sequentially decorated with mouse monoclonal antibody to the glucocorticoid receptor (panel A); mouse monoclonal antibody to poly(ADP-ribose) polymerase (panel B); rabbit antiserum to topoisomerase I (panel C): human autoimmune antiserum (panel D); a fresh aliquot of monoclonal antibody to the glucocorticoid receptor (panel E), and a fresh aliquot of monoclonal antibody to poly(ADP-ribose) polymerase (panel F). Antibodies were removed between exposures as described under Materials and Methods. Duplicate blots (not previously erased) were simultaneously exposed to the same monoclonal antibodies (panels B’, E’, and F, respectively) to assessretention of antigenicity. After five erasures, poly(ADP-tibose) polymerase was readily detected (panel F) with little decrement in signal intensity (cf. panels F and F). In contrast, the epitope detected by the antiglucocorticoid receptor antibody showed a marked decrease in reactivity after four erasure procedures (cf. panels E and E’).
94
KAUFMANN,
EWING, AND SHAPER
To assessany deleterious effect of the erasing procedure on the immobilized polypeptides, duplicate strips of nitrocellulose which had never been erased were exposed to the primary and secondary antibodies in parallel at three steps (Figs. 3B’, 3E’, and 3F’). The epitope recognized by the monoclonal antipoly(ADP-ribose) polymerase antibody was detected with no decrement in signal strength after the piece of nitrocellulose had been erased once (cf. Figs. 3B’ and 3B) and only a modest decrement in signal strength after the piece of nitrocellulose had been erased five times (cf. Figs. 3F and 3F’). In contrast, the epitope recognized by the monoclonal antiglucocorticoid receptor antibody was difficult to detect after the piece of nitrocellulose had been erased four times (cf. Figs. 3E and 3E’).* Thus, some epitopes appear to be susceptible to destruction during the erasing procedure while others are highly resistant.
Application of Erasure Method to Nylon Blots Because of its durability, nylon is being used increasingly in place of nitrocellulose as a solid support for immobilizing polypeptides. To explore the possibility of reusing blots after immobilization of polypeptides on nylon, we performed experiments similar to those presented in Fig. 1 (data not shown). Coating with milk prior to drying again facilitated complete removal of primary and secondary antibody by 2% SDS containing 100 mM P-mercaptoethanol at 70”C.3 When coating with milk was omitted prior to drying and autoradiography, small amounts of primary antibody remained on the blot and * Subsequent blotting with polyclonal anti-receptor antibody has confirmed that the 94 kD polypeptide is present on the nitrocellulose and has not been released by repeated erasure. 3Treatment for 30 min at 70°C with 3 M sodium thiocyanate containing 100 mM &mercaptoethanol and 62.5 mM Tris-HCI (pH 6.8 at 20°C) has also been shown in preliminary experiments to be capable of serving as an erasure buffer when polypeptides are immobilized on nylon.
could be detected during subsequent exposure to radiolabeled secondary antibody. Based upon these observations, we developed a modified method which allows reuse of protein blots when polypeptides have been immobilized on nylon (see Materials and Methods). DISCUSSION
We have devised a technique which removes antibodies from Western blots and thereby permits reuse of protein samples immobilized on nitrocellulose. The essential features of this technique are (i) recoating of blots with 5% (w/v) milk prior to drying and (ii) solubilization of antibodies at 70°C with 2% SDS containing 100 mM P-mercaptoethanol. The mechanism by which recoating nitrocellulose blots with milk facilitates the subsequent removal of antibodies is unknown. When blots were washed only with proteinfree buffer prior to drying, a substantial fraction of the bound antibody resisted solubilization under denaturing conditions (Figs. 1A and 2A, lane 2). This suggested the possibility of covalent attachment to the nitrocellulose or to nitrocellulose-immobilized proteins. Coating of blots with the reducing agent dithiothreitol or the antioxidant thiodiglycol prior to drying did not improve the subsequent solubilization of antibodies (Figs. 2B and 2C). On the other hand, coating of blots with bovine serum albumin, hemoglobin, or gelatin at commonly used concentrations improved subsequent removal of antibodies. Bovine milk, a convenient protein carrier in other immunological reactions (13,14) was even more effective than these other proteins at facilitating the erasure of Western blots. When blots were recoated with milk prior to drying, antibodies could be subsequently removed by treatment at 70°C for 30 min with buffered 2% SDS containing 100 mM /3-mercaptoethanol. Both SDS and reducing agent were required for complete solubilization of the antibodies (cf. lanes 2 and 3 in
THE ERASABLE
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95
In summary, the ability to sequentially Fig. 1B). Samples had to be heated to at least probe a single piece of nitrocellulose with 60°C in this buffer to remove all detectable radiolabel (data not shown). multiple antibodies has applicability in a wide range of situations (see introduction). The technique described above effectively solubilizes polyclonal antibodies from a vari- The technique described in the present communication should be useful in many laboraety of sources (Figs. 1 and 3). Our experience with the monoclonal antiglucocorticoid re- tories where Western blotting is performed. ceptor antibody (Fig. 3E) indicates that some epitopes will be damaged by the erasure proACKNOWLEDGMENTS cedure. On the other hand, the conditions We thank Drs. Sam Okret. J-A. Gustafsson, A-C. used to erase the blots (2% SDS containing Wikstriim, G. Poirier. L. Liu, and T. Provost for gener100 mM /3-mercaptoethanol, 70°C for 30 ously providing antibodies. Helpful discussions with min) are similar to conditions used to solu- Ralph Parchment and secretarial assistance by Sandy Smith are gratefully acknowledged. This work was supbilize polypeptides prior to electrophoresis. ported in part by grants from the NIH (GMl0518, Most epitopes which survive SDS-PAGE AM19300) and NSF (DCB-8417152). and electrophoretic transfer to nitrocellulose would be expected to survive the erasure REFERENCES procedure. Data obtained with monoclonal 1. Gershoni, J. M., and Palade, G. E. (1983) Anal. anti-poly(ADP-ribose) polymerase antibody Biochem. 131, l-15. (Figs. 3B and 3F) are consistent with this 2. Bers, G., and Garfin, D. (1985) BioTechniques 3, view. 276-288. The technique described in the present 3. Olmsted, J. B. (1981) J. Biol. CIrerem.256, 11955communication has been developed using 11957. 4. Smith, D. E., and Fisher, P. A. (1984) J. Cell Biol. radiolabeled secondary antibodies. It should 99,20-28. be equally applicable to the removal of col5. Earnshaw, W. C., and Rothfield, N. (1985) Chroloidal gold-labeled antibodies from Western mosoma 91, 3 13-32 1. blots ( 15,16). When alternative procedures 6. Kaufmann, S. H.. Gibson, W., and Shaper, J. H. are used to visualize antigen-antibody com(1983) J. Biol. Chem. 258, 2710-2719. 7. Kaufmann, S. H., and Shaper, J. H. (1984) Exp. plexes, the technique might have to be alCell Rex 155, 477-495. tered. For example, if silver enhancement 8. Okret, S., Wikstrom, A-C., Wrange, 0.. Andersson, techniques ( 15) or enzyme-mediated reacB., and Gustafsson. J-A. (1984) Proc. Null. Acad. tions (reviewed in (2)) are used to visualize Sci. CLSASI, 1609-1613. antigen-antibody complexes, additional 9. Lamarre, D., Talbot, B., Leduc, Y., Muller. S., and Poirier, G. (I 986) Biochern. Cell Biol. 64, steps might be needed to solubilize elemental 368-376. silver or enzyme reaction product, respec10. Fraker, P. J., and Speck, J. C., Jr. (I 978) Biochem. tively. Likewise, the technique might require Biophyr. Rex Commun. 80, 849-857. alteration when protein A is used to visualize 11. Legocki, R. P.. and Verma, D. P. S. (1981) Anal. antigen-antibody complexes. The IgG2 in Biochem. Ill, 385-392. 12. Erickson. P. F.. Minier, L. N., and Lasher, R. S. bovine milk binds protein A (17) albeit (1982) J. Irnmuno/. Methods 51, 241-249. weakly. As a result, blots on which nonspe13. Johnson, D. A., Gautsch. J. W.. Sportsman, J. R., cific protein binding sites are initially coated and Elder, J. H. (1984) Gene Anal. Tech. 1, 3-8. with milk exhibit a higher background level 14. Duhamel, R. C., and Johnson, D. A. (I 986) .I. Hisof protein A binding than blots initially tochem. Cytochem. 33, 71 l-l 14. coated with bovine serum albumin (data not 15. Moeremans. M., Daneels, G., Van Dijck, A., Langanger, G., and DeMey, J. (1984) J. Immunoi. shown). Consequently, when protein A is to Methods 74, 353-360. be used, exposure to milk probably should be 16. Hsu. Y-H. (1984) Anal. Biochem. 142,221-225. avoided until just before the blot is to be 17. Lindmark. R., Thoren-Tolling, K., and Sjoquist. J., dried for autoradiography. (1983) J. Immunol. Methods 62, 1-13.
BIOCHEMISTRY
161, 89-95
(1987)
The Erasable Western Blot SCOTTH.KAUFMANN,*CHARLES *Oncology
M. EWING,* ANDJOEL. H. SHAPER*.?
Center, Johns Hopkins Hospital and tDepartment Johns Hopkins University School of Medicine,
of Pharmacology and Molecular Baltimore, Mar.vland 21205
Sciences.
Received August 8, 1986 A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a lo-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70°C in 2% (w/v) sodium dodecyl sulfate containing 100 mM &mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed. o 1987 Academic press, IX. KEY WORDS: gel electrophoresis, protein: blotting: immunological methods, nuclear envelope polypeptides; antibodies.
Current methods of immobilizing electro- blots in order to reutilize the antibodies have phoretically separated polypeptides on deriv- been described (e.g., (3-5)), there is no atized paper (reviewed in (1,2)) permit the widely accepted method for removing antirapid analysis of multiple samples for the bodies from blots for the purpose of reusing presence of antigens of interest. There are the blots. In the present study we describe a several situations in which it would be conve- method for removing monoclonal and polynient to sequentially probe a single Western clonal antibodies from dried nitrocellulose blot with different antibodies: (i) when the blots while preserving the immobilized polyimmobilized polypeptides are derived from a peptides. A modification of this method also biological fluid or subcellular fraction which permits reuse of Western blots when proteins is available in small quantities and/or at are immobilized on nylon. great expense, (ii) when a purification scheme is to be monitored simultaneously MATERIALS AND METHODS for the presence of more than one antigen, (iii) when a large number of antibody soluMaterials. Sodium dodecyl sulfate, dithiotions (e.g., hybridoma supernatants or se- threitol, P-mercaptoethanol, and all chemiquential bleeds from a group of subjects) cals for electrophoresis were from Bio-Rad have to be assayed for the presence of anti- (Richmond, CA). Sartorius brand nitrocellubodies against a single antigen or class of an- lose paper (0.45~rcrn pore size) was from tigens, and (iv) when a blot gives an unex- Vangard (Neptune, NJ). Derivatized nylon pected result and probing with a second an- (GeneScreen) was from New England Nutibody will shed light on this result. clear (Boston, MA). Thiodiglycol was from While methods for eluting antibodies from Pierce (Rockford, IL). Bovine serum albu89
0003-2697187
$3.00
Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
90
KAUFMANN.
EWING,
min (RIA grade), bovine hemoglobin, and swine skin gelatin were from Sigma (St. Louis, MO). Goat anti-human IgG was from Tago (Burlingame, CA). Goat anti-rabbit IgG, rabbit anti-chicken IgG, and rabbit anti-mouse IgG were from Cappell (Cochranville, PA). Suflers. TS buffer: 10 mM Tris-HCl (pH 7.4 at 20°C) containing 150 mM NaCl. TSM buffer: TS buffer containing 5% (w/v) powdered nonfat milk, 1 mM sodium azide, 100 U/ml penicillin G, and 100 pg/ml streptomycin. Antibodies. Rat liver nuclear envelopes were prepared as previously described (6). Individual polypeptides separated by two-dimensional IEF’/SDS-PAGE (6) were used to immunize chickens according to a schedule previously described (7). The resulting antibodies will be characterized more completely in a subsequent paper. An IgG2, mouse monoclonal antibody against the 94-kDa steroid binding moiety of the rat liver glucocorticoid receptor (antibody No. 7 in Ref. (8)) was kindly provided by Drs. Sam Okret, Ann-Carlotte Wikstriim, and J-A. Gustafsson (Karolinska Institute). An IgG1 mouse monoclonal antibody against poly(ADP-ribose) polymerase (9) was kindly provided by Dr. Guy Poitier (University of Quebec). Rabbit polyclonal antiserum against bovine thymus topoisomerase I and human autoantibody against nuclear envelope polypeptides were kindly provided by Drs. Leroy Liu and Thomas Provost, respectively (Johns Hopkins University). Electrophoresis and transfer. Rat liver nuclei or nuclear envelopes were solubilized, reduced, and alkylated as previously described (6). Samples containing 2.5 X lo6 nuclei or 5 X lo6 nuclear envelopes were separated in the presense of SDS in 0.75-mmthick polyacrylamide gels containing 8% (w/v) acrylamide. Samples were electropho’ Abbreviations sodium dodecyl trophoresis.
used: IEF, isoelectric focusing; SDS, sulfate; PAGE, polyacrylamide gel elec-
AND
SHAPER
retically transferred to nitrocellulose at 4°C in a Hoefer series TE transfer apparatus operating at 90 V for 6- 12 h. Transfer buffer contained 20% (v/v) methanol, 192 mM glytine, 25 mM Tris, and 0.01% (w/v) SDS. All further steps were carried out at room temperature. Polypeptides transferred to nitrocellulose were visualized by staining with fast green FCF (CL 42053) and reequilibrated with TS buffer as previously described (7). The remaining protein binding sites were saturated during a 6- to 8-h incubation in TSM buffer. Incubation with antibodies prior to erasure. All steps were performed at room temperature. The sheets were incubated for 12- 14 h with antibodies diluted in TSM buffer. They were washed three times (15 min each) with TS buffer containing 2 M freshly deionized urea and 0.05% (w/v) Nonidet-P40 and then once (5 min) with TS buffer. Urea was omitted when monoclonal antibodies were used. The sheets were incubated for 60 min with TSM buffer containing 5-10 &i of lz51-labeled secondary antibody (labeled to a specific activity of 5-10 &i/pg by the method of Fraker and Speck (10)) and subjected to sequential 15-min washes (generally three or four) in TS buffer containing 0.05% (w/v) Nonidet-P40 until no radioactivity was detected in the wash buffer. Prior to drying, blots were incubated for 10 min in TSM buffer. They were then dried on absorbent paper at room temperature for 5 min and moved to fresh paper to complete the drying process. (Moving the coated blots after 5 min prevents their sticking to the paper.) They were exposed to Kodak XOmat XAR-5 or XRP-5 film at -70°C as indicated in the figure legends. Erasure of blots. Blots were incubated for 30 min at 70°C with erasure buffer consisting of 2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8 at 20”(Z), and 100 mM @-mercaptoethanol. All further steps were performed at room temperature. After two lo-min washes with TS buffer, the blots were dried (Figs. 1
THE
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and 2) or incubated with TSM buffer for 6 h prior to exposure to a new primary antibody (Fig. 3). Modijcation for use with nylon. With slight modification, the method described above can be used to remove primary and secondary antibodies from Western blots when proteins are immobilized on nylon rather than nitrocellulose. After SDS-PAGE, electrophoretic transfer was performed as described above. Staining with fast green FCF was omitted. The remaining protein binding sites were blocked by a 6- to S-h incubation at room temperature with 10% (w/v) powdered nonfat milk in TS buffer containing 1 mM sodium azide, 100 U/ml penicillin G, and 100 pg/ml streptomycin. After incubation with primary and secondary antibodies, blots were treated with TSM buffer for 10 min prior to drying and autoradiography. Primary and secondary antibodies were removed by a 30-min incubation at 70°C in erasure buffer. After two lo-min washes with TS buffer, blots were recoated for 6-8 h with 10% (w/v) powdered milk in the buffer described above and then exposed to a new primary antibody. RESULTS
D$kdty Erasing Dried Blots A number of investigators have described the elution of antibodies from protein blots using glycine at acid pH (e.g., (3,4)) or 3 M sodium thiocyanate at alkaline pH (5). In these procedures, blots were kept moist during all elution steps to preserve reactivity of the recovered antibodies. We were interested in the possibility of removing antibodies from nitrocellulose blots for the purpose of reusing the blots (e.g., (11,12). In this case, blots with their adherent antibodies were dried at ambient temperature and subjected to autoradiography prior to any attempted removal of the antibodies. When nitrocellulose blots were dried immediately after reaction with primary and secondary antibodies, subsequent treatment for 30 min at 70°C with 2% SDS containing
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100 mM @-mercaptoethanol, with 2% SDS containing 4 M urea, with 3 M sodium thiocyanate, with 0.2 M glycine-HCl (pH 2.5) or with 0.1 M sodium carbonate failed to remove all of the antibody (Fig. 1A, lanes 2-6, respectively). Likewise, treatment with 8 M urea containing 100 mM P-mercaptoethanol and 5 mg/ml bovine serum albumin at 60°C for 60 min (11) failed to remove all of the antibody (Fig. 1A, lane 7). Preliminary experiments also indicated difficulty in removing antibodies when blots had been coated with 3% bovine serum albumin rather than 5% milk prior to incubation with the primary antibody (data not shown). A fraction of the antibody remained tightly adherent to the nitrocellulose even after exposure to strongly denaturing conditions.
Coating with Protein prior to Drying Facilitates Erasure of Nitrocellulose Blots In an attempt to facilitate the subsequent removal of antibody, blots treated with primary and secondary antibody were recoated with 5% (w/v) powdered nonfat milk for 10 min prior to drying and autoradiography. This treatment did not detectably alter the amount of primary and secondary antibody bound to the blot (cf. lane 1, Figs. 1A and 1B). Subsequent treatment for 30 min at 70°C with 2% SDS containing 4 M urea, with 3 M sodium thiocyanate, with 0.2 M glycine (pH 2.5) or with 0.1 M sodium carbonate removed much more of the antibody than previously but still left a weakly detectable signal on the blot (Fig. 1B, lanes 4-7, respectively). Likewise, treatment with 8 M urea containing 100 mM B-mercaptoethanol and 5 mg/ml bovine serum albumin for 60 min at 60°C (11) left a weakly detectable signal (Fig. lB, lane 8). In contrast, treatment with 2% SDS containing 100 mM P-mercaptoethanol removed all of the detectable antibody (Fig. 1B, lane 2). To explore further the mechanism by which milk facilitated subsequent erasure of blots, replicate blots treated with primary and secondary antibody were coated with the
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EWING, AND SHAPER
quently treated with 2% SDS- 100 mM /3mercaptoethanol at 70°C for 30 min to remove antibodies (lane 2 in Figs. 2A-2G). Treatment with dithiothreitol (Fig. 2B) or * thiodiglycol (Fig. 2C) prior to drying did not 31 facilitate the subsequent removal of antibodies from blots. In contrast, treatment with commonly used concentrations of gelatin, B bovine serum albumin, or hemoglobin prior FIG. 1. Coating ofblots with milk prior to drying facihto drying improved subsequent removal of tates subsequent removal of antibodies. Replicate sam- antibody (Fig. 2, panels D-F, respectively). ples of rat liver nuclear envelopes were separated by SDS-PAGE and stained with Coomassie brilliant blue Nevertheless, these treatments left a weakly (lane 0) or transferred to nitrocellulose (panels A and B). detectable signal on the blots. Of the protein solutions tested, milk was most effective at After blocking of unoccupied binding sites in TSM buffer, a single piece of nitrocellulose was treated se- facilitating the subsequent removal of antiquentially with a I:5000 dilution of chicken serum con- body (Fig. 2G). 1234567
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taining antibodies directed against lamin B (upper arrowhead) followed by ‘251-labeled rabbit anti-chicken IgG. After appropriate washes (see Materials and Methods), pieces of the blot were incubated for IO min with TS buffer (panel A) or TSM buffer (panel B) immediately prior to drying. Autoradiography (not shown) confirmed the uniform labeling of lamin B in all lanes. Lanes were then excised and exposed for 30 min at 70°C to 62.5 mM Tris-HCI (pH 6.8 to 20°C) containing 2% (w/v) SDS and 100 mM @-mercaptoethanol (A2, B2, B9), 2% (w/v) SDS without reducing agent (B3), 2% (w/v) SDS and 4 M deionized urea (A3, B4), or 3 M sodium thiocyanate (A4, B5); to 0.2 M glycine-HC1 at pH 2.5 (AS, B6); to 0.1 M sodium carbonate (A6, B7): or for 60 min at 60°C to 8 M deionized urea containing 100 mM &mercaptoethanol and 5 mg/ml bovine serum albumin (A7, B8). After erasure, lane B9 was recoated with TSM buffer and treated sequentially with chicken serum containing antibodies directed against a 47-kDa nuclear envelope polypeptide (lower arrowhead) followed by ‘251labeled rabbit anti-chicken IgG. Lane 1 in each panel served as control. Autoradiography was for 16 days at -70°C with XRP-5 film. Nonadjacent lanes of a single autoradiograph have been juxtaposed to compose this figure. Note that recoating with milk prior to drying did not detectably alter the amount of antibody bound to the blot (cf. A 1 and Bl) but facilitated subsequent removal of antibody (panel B). In panel B all of the treatments except 2% SDS containing 100 mM P-mercaptoethanol (B2, B9) left a faintly detectable signal on the original autoradiograph.
reducing agent dithiothreitol, the antioxidant thiodiglycol, or with a number of different protein solutions prior to drying. None of these treatments detectably altered the amount of antibody bound to the blot (cf. lane 1 in Figs. 2A-2G). Blots were subse-
Repetitive
Use of a Single Blot
To assess the suitability of erased blots for subsequent detection of antigens, a lane
A
BCDEFG
FIG. 2. Effect of various coating solutions on the subsequent removal of antibodies. Replicate samples of rat liver nuclear envelopes on a single sheet of nitrocellulose were treated sequentially with a 1:5000 dilution of chicken serum containing antibodies to lamin B followed by ‘251-labeled anti-chicken IgG. Immediately prior to drying, pieces of nitrocellulose were treated for 10 min with TS buffer (panel A) or TS buffer containing 1 mM dithiothreitol (panel B), 1% (w/v) thiodiglycol (panel C), 0.1% (w/v) gelatin (panel D), 3% (w/v) bovine serum albumin (panel E), 1% (w/v) hemoglobin (panel F), or 5% (w/v) milk (panel G). Autoradiography (not shown) confirmed the uniform labeling of lamin B in all lanes. In each panel, lane 2 was incubated at 70°C with erasure buffer as described under Materials and Methods. Lane 1 served as a control. Autoradiography was for I6 days at -70°C with XRP-5 film. Nonadjacent panels from a single autoradiograph have been juxtaposed to compose this figure. Recoating with any protein solution facilitates subsequent removal of antibodies (panels D-G) but the removal is most complete when blots are treated with 5% (w/v) milk prior to drying (panel G).
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erased in 2% SDS containing 100 mM j3mercaptoethanol was recoated with milk and then reacted sequentially with another primary antibody (raised in the same species as the original antibody) followed by radiolabeled secondary antibody (Fig. lB, lane 9). The second antigen was readily detected. In addition, the lack of binding of radiolabel in the region where the original primary antibody was bound confirmed that treatment with 2% SDS containing 100 mM @-mercaptoethanol removed primary antibody as well as radiolabeled secondary antibody. To explore further, the repetitive use of a single blot, polypeptides from the livers of adrenalectomized and glucocorticoid-treated rats were separated by SDS-PAGE, transferred to nitrocellulose, and then reacted sequentially with the following antibodies and their corresponding radiolabeled secondary antibodies: mouse monoclonal antibody to the 94-kDa polypeptide moiety of the rat liver glucocorticoid receptor (Fig. 3A), mouse monoclonal antibody to poly(ADP-
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ribose) polymerase (Fig. 3B), rabbit polyclonal antibody to topoisomerase I (Fig. 3C), human polyclonal antibody to nuclear envelope polypeptides (Fig. 3D), and then fresh aliquots of the two mouse monoclonal antibodies (Figs. 3E and 3F). The results of this experiment are shown in Fig. 3. Consistent with current models of steroid hormone action, the glucocorticoid receptor was detectable in isolated nuclei from hormone-treated rats (Fig. 3A, right lane) but not adrenalectomized rats (Fig. 3A, left lane). The observation that other antibodies bind almost equally to both lanes (Figs. 3B-3D) indicates that the lack of binding of antiglucocorticoid receptor antibody to the sample in the left lane is due to selective absence of this particular antigen rather than aberrant loading or transfer of the polypeptides, thus illustrating one of the potential uses of this method. In addition, these results indicate that the erasing procedure described above permitted removal of various polyclonal antibodies as well as the two monoclonal antibodies.
.
FIG. 3. Repetitive use of a single piece of nitrocellulose. Nuclei from the livers of adrenalectamized (left lane) or glucocorticoid-treated (right lane) rats were subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose. After coating with TSM buffer, a single piece of nitrocellulose was sequentially decorated with mouse monoclonal antibody to the glucocorticoid receptor (panel A); mouse monoclonal antibody to poly(ADP-ribose) polymerase (panel B); rabbit antiserum to topoisomerase I (panel C): human autoimmune antiserum (panel D); a fresh aliquot of monoclonal antibody to the glucocorticoid receptor (panel E), and a fresh aliquot of monoclonal antibody to poly(ADP-ribose) polymerase (panel F). Antibodies were removed between exposures as described under Materials and Methods. Duplicate blots (not previously erased) were simultaneously exposed to the same monoclonal antibodies (panels B’, E’, and F, respectively) to assessretention of antigenicity. After five erasures, poly(ADP-tibose) polymerase was readily detected (panel F) with little decrement in signal intensity (cf. panels F and F). In contrast, the epitope detected by the antiglucocorticoid receptor antibody showed a marked decrease in reactivity after four erasure procedures (cf. panels E and E’).
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EWING, AND SHAPER
To assessany deleterious effect of the erasing procedure on the immobilized polypeptides, duplicate strips of nitrocellulose which had never been erased were exposed to the primary and secondary antibodies in parallel at three steps (Figs. 3B’, 3E’, and 3F’). The epitope recognized by the monoclonal antipoly(ADP-ribose) polymerase antibody was detected with no decrement in signal strength after the piece of nitrocellulose had been erased once (cf. Figs. 3B’ and 3B) and only a modest decrement in signal strength after the piece of nitrocellulose had been erased five times (cf. Figs. 3F and 3F’). In contrast, the epitope recognized by the monoclonal antiglucocorticoid receptor antibody was difficult to detect after the piece of nitrocellulose had been erased four times (cf. Figs. 3E and 3E’).* Thus, some epitopes appear to be susceptible to destruction during the erasing procedure while others are highly resistant.
Application of Erasure Method to Nylon Blots Because of its durability, nylon is being used increasingly in place of nitrocellulose as a solid support for immobilizing polypeptides. To explore the possibility of reusing blots after immobilization of polypeptides on nylon, we performed experiments similar to those presented in Fig. 1 (data not shown). Coating with milk prior to drying again facilitated complete removal of primary and secondary antibody by 2% SDS containing 100 mM P-mercaptoethanol at 70”C.3 When coating with milk was omitted prior to drying and autoradiography, small amounts of primary antibody remained on the blot and * Subsequent blotting with polyclonal anti-receptor antibody has confirmed that the 94 kD polypeptide is present on the nitrocellulose and has not been released by repeated erasure. 3Treatment for 30 min at 70°C with 3 M sodium thiocyanate containing 100 mM &mercaptoethanol and 62.5 mM Tris-HCI (pH 6.8 at 20°C) has also been shown in preliminary experiments to be capable of serving as an erasure buffer when polypeptides are immobilized on nylon.
could be detected during subsequent exposure to radiolabeled secondary antibody. Based upon these observations, we developed a modified method which allows reuse of protein blots when polypeptides have been immobilized on nylon (see Materials and Methods). DISCUSSION
We have devised a technique which removes antibodies from Western blots and thereby permits reuse of protein samples immobilized on nitrocellulose. The essential features of this technique are (i) recoating of blots with 5% (w/v) milk prior to drying and (ii) solubilization of antibodies at 70°C with 2% SDS containing 100 mM P-mercaptoethanol. The mechanism by which recoating nitrocellulose blots with milk facilitates the subsequent removal of antibodies is unknown. When blots were washed only with proteinfree buffer prior to drying, a substantial fraction of the bound antibody resisted solubilization under denaturing conditions (Figs. 1A and 2A, lane 2). This suggested the possibility of covalent attachment to the nitrocellulose or to nitrocellulose-immobilized proteins. Coating of blots with the reducing agent dithiothreitol or the antioxidant thiodiglycol prior to drying did not improve the subsequent solubilization of antibodies (Figs. 2B and 2C). On the other hand, coating of blots with bovine serum albumin, hemoglobin, or gelatin at commonly used concentrations improved subsequent removal of antibodies. Bovine milk, a convenient protein carrier in other immunological reactions (13,14) was even more effective than these other proteins at facilitating the erasure of Western blots. When blots were recoated with milk prior to drying, antibodies could be subsequently removed by treatment at 70°C for 30 min with buffered 2% SDS containing 100 mM /3-mercaptoethanol. Both SDS and reducing agent were required for complete solubilization of the antibodies (cf. lanes 2 and 3 in
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In summary, the ability to sequentially Fig. 1B). Samples had to be heated to at least probe a single piece of nitrocellulose with 60°C in this buffer to remove all detectable radiolabel (data not shown). multiple antibodies has applicability in a wide range of situations (see introduction). The technique described above effectively solubilizes polyclonal antibodies from a vari- The technique described in the present communication should be useful in many laboraety of sources (Figs. 1 and 3). Our experience with the monoclonal antiglucocorticoid re- tories where Western blotting is performed. ceptor antibody (Fig. 3E) indicates that some epitopes will be damaged by the erasure proACKNOWLEDGMENTS cedure. On the other hand, the conditions We thank Drs. Sam Okret. J-A. Gustafsson, A-C. used to erase the blots (2% SDS containing Wikstriim, G. Poirier. L. Liu, and T. Provost for gener100 mM /3-mercaptoethanol, 70°C for 30 ously providing antibodies. Helpful discussions with min) are similar to conditions used to solu- Ralph Parchment and secretarial assistance by Sandy Smith are gratefully acknowledged. This work was supbilize polypeptides prior to electrophoresis. ported in part by grants from the NIH (GMl0518, Most epitopes which survive SDS-PAGE AM19300) and NSF (DCB-8417152). and electrophoretic transfer to nitrocellulose would be expected to survive the erasure REFERENCES procedure. Data obtained with monoclonal 1. Gershoni, J. M., and Palade, G. E. (1983) Anal. anti-poly(ADP-ribose) polymerase antibody Biochem. 131, l-15. (Figs. 3B and 3F) are consistent with this 2. Bers, G., and Garfin, D. (1985) BioTechniques 3, view. 276-288. The technique described in the present 3. Olmsted, J. B. (1981) J. Biol. CIrerem.256, 11955communication has been developed using 11957. 4. Smith, D. E., and Fisher, P. A. (1984) J. Cell Biol. radiolabeled secondary antibodies. It should 99,20-28. be equally applicable to the removal of col5. Earnshaw, W. C., and Rothfield, N. (1985) Chroloidal gold-labeled antibodies from Western mosoma 91, 3 13-32 1. blots ( 15,16). When alternative procedures 6. Kaufmann, S. H.. Gibson, W., and Shaper, J. H. are used to visualize antigen-antibody com(1983) J. Biol. Chem. 258, 2710-2719. 7. Kaufmann, S. H., and Shaper, J. H. (1984) Exp. plexes, the technique might have to be alCell Rex 155, 477-495. tered. For example, if silver enhancement 8. Okret, S., Wikstrom, A-C., Wrange, 0.. Andersson, techniques ( 15) or enzyme-mediated reacB., and Gustafsson. J-A. (1984) Proc. Null. Acad. tions (reviewed in (2)) are used to visualize Sci. CLSASI, 1609-1613. antigen-antibody complexes, additional 9. Lamarre, D., Talbot, B., Leduc, Y., Muller. S., and Poirier, G. (I 986) Biochern. Cell Biol. 64, steps might be needed to solubilize elemental 368-376. silver or enzyme reaction product, respec10. Fraker, P. J., and Speck, J. C., Jr. (I 978) Biochem. tively. Likewise, the technique might require Biophyr. Rex Commun. 80, 849-857. alteration when protein A is used to visualize 11. Legocki, R. P.. and Verma, D. P. S. (1981) Anal. antigen-antibody complexes. The IgG2 in Biochem. Ill, 385-392. 12. Erickson. P. F.. Minier, L. N., and Lasher, R. S. bovine milk binds protein A (17) albeit (1982) J. Irnmuno/. Methods 51, 241-249. weakly. As a result, blots on which nonspe13. Johnson, D. A., Gautsch. J. W.. Sportsman, J. R., cific protein binding sites are initially coated and Elder, J. H. (1984) Gene Anal. Tech. 1, 3-8. with milk exhibit a higher background level 14. Duhamel, R. C., and Johnson, D. A. (I 986) .I. Hisof protein A binding than blots initially tochem. Cytochem. 33, 71 l-l 14. coated with bovine serum albumin (data not 15. Moeremans. M., Daneels, G., Van Dijck, A., Langanger, G., and DeMey, J. (1984) J. Immunoi. shown). Consequently, when protein A is to Methods 74, 353-360. be used, exposure to milk probably should be 16. Hsu. Y-H. (1984) Anal. Biochem. 142,221-225. avoided until just before the blot is to be 17. Lindmark. R., Thoren-Tolling, K., and Sjoquist. J., dried for autoradiography. (1983) J. Immunol. Methods 62, 1-13.