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Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes
Journal oflmmunologicalMethods, 81 (1985)161-165 Elsevier
161
JIM03550
Short Communication
Different Blocking Agents Cause Variation in the Immunologic Detection of Proteins Transferred to Nitrocellulose Membranes S t a n l e y M. Spinola i a n d J a n n e G . ' C a n n o n 2 Departments of ~ Pediatrics and 2 Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. (Received 24 December 1984, accepted 11 March 1985)
We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins. Key words: Western blotting - blocking agents
Introduction Several methods describing the transfer of electrophoretically separated proteins to nitrocellulose membranes for immunologic detection, or 'Western blotting', have been published (Towbin et al., 1979; Burnette, 1981; Towbin and Gordon, 1984). In some methods a protein such as bovine serum albumin or gelatin is employed as a blocking agent to saturate free protein binding sites on the nitrocellulose membrane prior to probing it with antibody. Such treatment should reduce non-specific binding of antibody to the nitrocellulose while allowing detection of immunologically reactive protein bands when antigen antibody complexes are identified with an agent such as radiolabeled staphylococcal protein A or anti-immunoglobulin antibody. Washing nitrocellulose with non-ionic detergents such as Nonidet P-40 is also useful in reducing non-specific protein binding (Burnette, 1981), and may aid in renaturation of antigenic sites (Petit et al., 1982). Recently Western. blotting procedures employing Tween 20 (Batteiger et al., 1982) and non-fat dry milk with Antifoam (Johnson et al., 1984) as blocking agents and in Abbreviations: BSA, bovine serum albumin; NP-40, Nonidet P-40; SDS, sodium dodecyl sulfate; TS, Tris saline. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
162 washing solutions have been described. The former method is advantageous in that it allows staining of nitrocellulose for transferred proteins after immunologic probing and assaying for bound antibody. The latter method dramatically reduces nonspecific antibody binding; both methods are inexpensive. The purpose of this study was to compare BSA, non-fat dry milk and Tween 20 as blocking agents in a Western blotting procedure involving the detection of bacterial antigens.
Materials and Methods
Outer membranes of a non-typable Haemophilus influenzae nasopharyngeal isolate, K71-28 (obtained from Dr. Floyd Denny), were prepared by a Sarkosyl (Geigy Chemicals) extraction method (Barenkamp et al., 1981). Cells of Neisseria gonorrhoeae strain FA1090 (Nachamkin et al., 1981) were scraped from agar plates. Antigens were solubilized at 100°C for 5 rain in 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.125 M Tris, pH 6.8. Proteins were separated by SDS-polyacrylamide gel electrophoresis, using a linear acrylamide gradient (4%-30%) and the discontinuous buffer system of Laemmli (1970). Electrophoresis was at 200 V (constant voltage) at 4°C for 24 h. All reagents for electrophoresis were from Bio-Rad Laboratories. Western transfer of electrophoretically separated proteins to nitrocellulose (BA85; Schleicher and Schuell) was done in a Transblot apparatus (Bio-Rad Laboratories) at 8 V/cm according to the method of Burnette (1981). Sera were prepared in the following manner: a rabbit was immunized by repeated subcutaneous injection at 30 day intervals of outer membranes of H. influenzae K71-28. Serum from the child from whom isolate K71-28 was cultured was provided by Dr. Floyd Denny. A 1:5000 dilution of the rabbit hyperimmune serum and a 1:1000 dilution of the human serum was used for immunologic probing of the Haemophilus outer membranes. Two mice were immunized by intraperitoneal injections of outer membranes of N. gonorrhoeae strain FA1090, on days 1 and 14, and their serum was pooled. Monoclonal antibody H.8, which binds to a common Neisseria antigen with an apparent MW of 20,000, has been previously described (Cannon et al., 1984). A 1 : 500 dilution of the mouse polyclonal serum and a 1 : 15 dilution of H.8 culture supernatant were used for probing of gonococcal proteins. Transferred proteins were probed by a modification of a method of Hu et al. (1981). Nitrocellulose membranes were cut into vertical 5 mm strips and placed in 15 ml polystyrene tubes. The strips were incubated with 5 ml of a solution (see Fig. 1 for details) containing a blocking agent at 37°C for 2 h on a rotating platform. Subsequent steps were done at room temperature. The strips were incubated in 2 ml of a solution containing diluted serum for 1 h. Controls were incubated in solutions lacking serum. Strips were washed 4 times with 5 ml of an appropriate buffer for 15 min for each change, incubated with 1/~Ci of radioiodinated protein A (Amersham) in 2 ml of appropriate solution for 30 min, and washed 4 times as above. Strips were dried at 37°C for 1 h and autoradiographed. Each experiment was done in triplicate.
163
Results and Discussion
Results of probing outer membrane proteins of the non-typable H. influenzae isolate with rabbit hyperimmune serum are shown in Fig. 1, panel A. When BSA (lane 1) was used as a blocking agent and TS was used in washing, numerous bands were detected but several of the bands were difficult to distinguish from background. The addition of NP-40 to the washing solution (lane 2) decreased background, allowing the detection of several minor bands which were not clearly seen when TS was used in washing. The addition of NP-40 also caused the loss of 1 major band and decreased intensity of several other bands seen in lane 1. When Tween 20 (lane 3) was used as a blocking agent 1 additional major band was seen and several bands were intensified. Commercially obtained non-fat dry milk dramatically reduced background but caused the loss of numerous bands (lane 4)."There was no further loss of bands when Antifoam was used in the non-fat dry milk solution (lane 5). Similar results were seen when Haemophilus antigens were probed with human serum (Fig. 1, panel B) except that no bands were detected when BSA was used in blocking and TS was used in washing because of intense background (lane 1). Controls (not shown) showed no immunologically reactive bands under all conditions. We obtained similar results when gonococcal proteins were probed with mouse polyclonal serum (Fig. 1, panel C). When BSA was used as a blocking agent and TS was used in washing (lane 1), there were numerous bands detected, with acceptable background. The addition of NP-40 to the washing solution (lane 2) eliminated several of these signals as did non-fat dry milk (lane 4) with Antifoam (lane 5). The Tween 20 procedure (lane 3) caused changes in relative intensity of several bands, and the appearance of another band which differed in position from those seen in lane 1. Controls (not shown) did not demonstrate any immunologically reactive proteins under all conditions. We noted qualitatively different results when monoclonal antibody H.8 was used to probe gonococcal proteins (Fig. 1, panel D). Binding of H.8 to its antigen was detected only when BSA was used as a blocking agent and TS was used in washing (lane 1). All other test conditions disrupted detection of this reaction. In all of these experiments, there were discrepancies in the detection of immunologically reactive proteins by the Western blotting procedure that depended on the blocking and washing agents used. When polyclonal sera were used as probes, Tween 20 generally allowed identification of the greatest number of antigen antibody complexes. In 2 cases washing BSA-blocked strips with NP-40 diminished background and allowed the detection of several bands not clearly seen when BSA blocked strips were washed in buffer alone. In 1 case, however, blocking with BSA and washing with buffer alone gave good resolution of bands and further washing with NP-40 eliminated half of the signals. Non-fat dry milk reduced background but also drastically reduced the number of antigen complexes seen. This effect did not appear to be due to Antifoam, which is included in the procedure described by Johnson et al. (1984). When a monoclonal antibody was used as a probe, the detection of an antigen antibody complex was possible only when BSA was used as a blocking agent and TS was used alone in washing.
164
B
A 1 2 345
C 1 2345
D 12345
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Fig. 1. Panel A. Autoradiograrn comparing the effect of different blocking, diluent and wash reagents on probing outer membrane proteins of a non-typable Haemophilus influenzae isolate with rabbit hyperimmune serum. Lane 1: "Iris saline-bovine serum albumin used as a blocking agent and as carrier solution during antibody probing and incubation with 125I-protein A; TS used in washes. Lane 2: as in lane 1 but TS-NP-40 used in 2 middle washes of both washing steps. Lane 3: TS-Tween used throughout. Lane 4: TS-non-fat dry milk used throughout. Lane 5: TS-non-fat dry milk with Antifoam used throughout. Autoradiogram was developed after 48 h of exposure. Panel B. Effects of the same treatments on probing Haemophilus antigens with human serum. The treatment of the strips in lanes 1-5 was the same as in the corresponding lanes of panel A. Autoradiogram developed after 5 days of exposure. Panel C. Effect of the same treatments on probing gonococcal proteins with mouse hyperimmune serum. Treatment in lanes 1-5 corresponds with panel A. Autoradiogram was developed after 4 days of exposure. Panel D. Effect of the same treatments on probing gonococcal proteins with monoclonal antibody H.8. Treatment in lanes 1-5 corresponds with panel A. Autoradiogram was developed after 5 days of exposure. Reagents were prepared as follows: TS, 10 mM tris, 0.9% NaC1, pH 7.4; TS-BSA, TS containing 5% (w/v) BSA; TS-Tween, TS containing 0.05% (v/v) Tween 20, TS-NP-40, TS containing 0.05% (v/v) NP-40; TS-non-fat dry milk, TS containing 5% w / v non-fat dry milk (Carnation); TS-non-fat dry milk with Antifoam (Sigma), TS containing 5% (w/v) non-fat dry milk with 0.01% v / v Antifoam. T h e c a u s e o f t h e s e d i s c r e p a n t results was u n c l e a r . B a n d s m a y h a v e b e e n lost b e c a u s e a n t i g e n fell o f f n i t r o c e l l u l o s e , b e c a u s e o f d i s r u p t i o n o f a n t i g e n a n t i b o d y c o m p l e x e s b y a r e a g e n t o r b e c a u s e o f n o n - s p e c i f i c b i n d i n g o f r e a g e n t s to a n t i g e n o r antibody. Detection of other bands may have been enhanced by renaturation of
165
epitopes by non-ionic detergent. It was not possible to say what was occurring with each agent. We conclude that several methods of blocking and washing need to be compared when probing antigens by the Western blotting procedure to determine if otherwise detectable antigen antibody complexes are disrupted by the reagents used. Acknowledgements This research was supported b y Public Health Service Grants AI15036 and AI07151-06 from the National Institute of Allergy and Infectious Diseases and HL19171 from the National Heart, Lung and Blood Institute. We thank Anne Stephenson for her assistance with these studies, and Lynn Brooks for preparation of the manuscript.
References Barenkamp, S.J., R.S. Munson and D.M. Granoff, 1981, J. Infect. Dis. 143, 668. Batteiger, B., W.J. Newhall V and R.B. Jones, 1982, J. Immunol. Methods 55, 297. Burnette, W.N., 1981, Affal. Biochem. 112, 195. Cannon, J.G., W.J. Black, I. Nachamkin and P.W. Stewart, 1984, Infect. Immun. 43, 994. Hu, P.C., Y. Huang, J.A. Graham and D.E. Gardner, 1981, Biochem. Biophys. Res. Commun. 103, 1963. Johnson, D.A., J.W. Gautsch, J.R. Sportsman and J. Elder, 1984, Gene Anal. Techn. 1, 3. Laemmli, U.K., 1970, Nature (London) 227, 680. Nachamkin, I., J.G. Cannon and R.S. Mittler, 1981, Infect. Immun. 32, 641. Petit~ C., M.E. Sauron and J. Theze, 1982, Ann. Immunol. 133D, 77. Towbin, H. and J. Gordon, 1984, J. Immunol. Methods 72, 313. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350.
161
JIM03550
Short Communication
Different Blocking Agents Cause Variation in the Immunologic Detection of Proteins Transferred to Nitrocellulose Membranes S t a n l e y M. Spinola i a n d J a n n e G . ' C a n n o n 2 Departments of ~ Pediatrics and 2 Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. (Received 24 December 1984, accepted 11 March 1985)
We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins. Key words: Western blotting - blocking agents
Introduction Several methods describing the transfer of electrophoretically separated proteins to nitrocellulose membranes for immunologic detection, or 'Western blotting', have been published (Towbin et al., 1979; Burnette, 1981; Towbin and Gordon, 1984). In some methods a protein such as bovine serum albumin or gelatin is employed as a blocking agent to saturate free protein binding sites on the nitrocellulose membrane prior to probing it with antibody. Such treatment should reduce non-specific binding of antibody to the nitrocellulose while allowing detection of immunologically reactive protein bands when antigen antibody complexes are identified with an agent such as radiolabeled staphylococcal protein A or anti-immunoglobulin antibody. Washing nitrocellulose with non-ionic detergents such as Nonidet P-40 is also useful in reducing non-specific protein binding (Burnette, 1981), and may aid in renaturation of antigenic sites (Petit et al., 1982). Recently Western. blotting procedures employing Tween 20 (Batteiger et al., 1982) and non-fat dry milk with Antifoam (Johnson et al., 1984) as blocking agents and in Abbreviations: BSA, bovine serum albumin; NP-40, Nonidet P-40; SDS, sodium dodecyl sulfate; TS, Tris saline. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
162 washing solutions have been described. The former method is advantageous in that it allows staining of nitrocellulose for transferred proteins after immunologic probing and assaying for bound antibody. The latter method dramatically reduces nonspecific antibody binding; both methods are inexpensive. The purpose of this study was to compare BSA, non-fat dry milk and Tween 20 as blocking agents in a Western blotting procedure involving the detection of bacterial antigens.
Materials and Methods
Outer membranes of a non-typable Haemophilus influenzae nasopharyngeal isolate, K71-28 (obtained from Dr. Floyd Denny), were prepared by a Sarkosyl (Geigy Chemicals) extraction method (Barenkamp et al., 1981). Cells of Neisseria gonorrhoeae strain FA1090 (Nachamkin et al., 1981) were scraped from agar plates. Antigens were solubilized at 100°C for 5 rain in 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.125 M Tris, pH 6.8. Proteins were separated by SDS-polyacrylamide gel electrophoresis, using a linear acrylamide gradient (4%-30%) and the discontinuous buffer system of Laemmli (1970). Electrophoresis was at 200 V (constant voltage) at 4°C for 24 h. All reagents for electrophoresis were from Bio-Rad Laboratories. Western transfer of electrophoretically separated proteins to nitrocellulose (BA85; Schleicher and Schuell) was done in a Transblot apparatus (Bio-Rad Laboratories) at 8 V/cm according to the method of Burnette (1981). Sera were prepared in the following manner: a rabbit was immunized by repeated subcutaneous injection at 30 day intervals of outer membranes of H. influenzae K71-28. Serum from the child from whom isolate K71-28 was cultured was provided by Dr. Floyd Denny. A 1:5000 dilution of the rabbit hyperimmune serum and a 1:1000 dilution of the human serum was used for immunologic probing of the Haemophilus outer membranes. Two mice were immunized by intraperitoneal injections of outer membranes of N. gonorrhoeae strain FA1090, on days 1 and 14, and their serum was pooled. Monoclonal antibody H.8, which binds to a common Neisseria antigen with an apparent MW of 20,000, has been previously described (Cannon et al., 1984). A 1 : 500 dilution of the mouse polyclonal serum and a 1 : 15 dilution of H.8 culture supernatant were used for probing of gonococcal proteins. Transferred proteins were probed by a modification of a method of Hu et al. (1981). Nitrocellulose membranes were cut into vertical 5 mm strips and placed in 15 ml polystyrene tubes. The strips were incubated with 5 ml of a solution (see Fig. 1 for details) containing a blocking agent at 37°C for 2 h on a rotating platform. Subsequent steps were done at room temperature. The strips were incubated in 2 ml of a solution containing diluted serum for 1 h. Controls were incubated in solutions lacking serum. Strips were washed 4 times with 5 ml of an appropriate buffer for 15 min for each change, incubated with 1/~Ci of radioiodinated protein A (Amersham) in 2 ml of appropriate solution for 30 min, and washed 4 times as above. Strips were dried at 37°C for 1 h and autoradiographed. Each experiment was done in triplicate.
163
Results and Discussion
Results of probing outer membrane proteins of the non-typable H. influenzae isolate with rabbit hyperimmune serum are shown in Fig. 1, panel A. When BSA (lane 1) was used as a blocking agent and TS was used in washing, numerous bands were detected but several of the bands were difficult to distinguish from background. The addition of NP-40 to the washing solution (lane 2) decreased background, allowing the detection of several minor bands which were not clearly seen when TS was used in washing. The addition of NP-40 also caused the loss of 1 major band and decreased intensity of several other bands seen in lane 1. When Tween 20 (lane 3) was used as a blocking agent 1 additional major band was seen and several bands were intensified. Commercially obtained non-fat dry milk dramatically reduced background but caused the loss of numerous bands (lane 4)."There was no further loss of bands when Antifoam was used in the non-fat dry milk solution (lane 5). Similar results were seen when Haemophilus antigens were probed with human serum (Fig. 1, panel B) except that no bands were detected when BSA was used in blocking and TS was used in washing because of intense background (lane 1). Controls (not shown) showed no immunologically reactive bands under all conditions. We obtained similar results when gonococcal proteins were probed with mouse polyclonal serum (Fig. 1, panel C). When BSA was used as a blocking agent and TS was used in washing (lane 1), there were numerous bands detected, with acceptable background. The addition of NP-40 to the washing solution (lane 2) eliminated several of these signals as did non-fat dry milk (lane 4) with Antifoam (lane 5). The Tween 20 procedure (lane 3) caused changes in relative intensity of several bands, and the appearance of another band which differed in position from those seen in lane 1. Controls (not shown) did not demonstrate any immunologically reactive proteins under all conditions. We noted qualitatively different results when monoclonal antibody H.8 was used to probe gonococcal proteins (Fig. 1, panel D). Binding of H.8 to its antigen was detected only when BSA was used as a blocking agent and TS was used in washing (lane 1). All other test conditions disrupted detection of this reaction. In all of these experiments, there were discrepancies in the detection of immunologically reactive proteins by the Western blotting procedure that depended on the blocking and washing agents used. When polyclonal sera were used as probes, Tween 20 generally allowed identification of the greatest number of antigen antibody complexes. In 2 cases washing BSA-blocked strips with NP-40 diminished background and allowed the detection of several bands not clearly seen when BSA blocked strips were washed in buffer alone. In 1 case, however, blocking with BSA and washing with buffer alone gave good resolution of bands and further washing with NP-40 eliminated half of the signals. Non-fat dry milk reduced background but also drastically reduced the number of antigen complexes seen. This effect did not appear to be due to Antifoam, which is included in the procedure described by Johnson et al. (1984). When a monoclonal antibody was used as a probe, the detection of an antigen antibody complex was possible only when BSA was used as a blocking agent and TS was used alone in washing.
164
B
A 1 2 345
C 1 2345
D 12345
12345 i
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i
Fig. 1. Panel A. Autoradiograrn comparing the effect of different blocking, diluent and wash reagents on probing outer membrane proteins of a non-typable Haemophilus influenzae isolate with rabbit hyperimmune serum. Lane 1: "Iris saline-bovine serum albumin used as a blocking agent and as carrier solution during antibody probing and incubation with 125I-protein A; TS used in washes. Lane 2: as in lane 1 but TS-NP-40 used in 2 middle washes of both washing steps. Lane 3: TS-Tween used throughout. Lane 4: TS-non-fat dry milk used throughout. Lane 5: TS-non-fat dry milk with Antifoam used throughout. Autoradiogram was developed after 48 h of exposure. Panel B. Effects of the same treatments on probing Haemophilus antigens with human serum. The treatment of the strips in lanes 1-5 was the same as in the corresponding lanes of panel A. Autoradiogram developed after 5 days of exposure. Panel C. Effect of the same treatments on probing gonococcal proteins with mouse hyperimmune serum. Treatment in lanes 1-5 corresponds with panel A. Autoradiogram was developed after 4 days of exposure. Panel D. Effect of the same treatments on probing gonococcal proteins with monoclonal antibody H.8. Treatment in lanes 1-5 corresponds with panel A. Autoradiogram was developed after 5 days of exposure. Reagents were prepared as follows: TS, 10 mM tris, 0.9% NaC1, pH 7.4; TS-BSA, TS containing 5% (w/v) BSA; TS-Tween, TS containing 0.05% (v/v) Tween 20, TS-NP-40, TS containing 0.05% (v/v) NP-40; TS-non-fat dry milk, TS containing 5% w / v non-fat dry milk (Carnation); TS-non-fat dry milk with Antifoam (Sigma), TS containing 5% (w/v) non-fat dry milk with 0.01% v / v Antifoam. T h e c a u s e o f t h e s e d i s c r e p a n t results was u n c l e a r . B a n d s m a y h a v e b e e n lost b e c a u s e a n t i g e n fell o f f n i t r o c e l l u l o s e , b e c a u s e o f d i s r u p t i o n o f a n t i g e n a n t i b o d y c o m p l e x e s b y a r e a g e n t o r b e c a u s e o f n o n - s p e c i f i c b i n d i n g o f r e a g e n t s to a n t i g e n o r antibody. Detection of other bands may have been enhanced by renaturation of
165
epitopes by non-ionic detergent. It was not possible to say what was occurring with each agent. We conclude that several methods of blocking and washing need to be compared when probing antigens by the Western blotting procedure to determine if otherwise detectable antigen antibody complexes are disrupted by the reagents used. Acknowledgements This research was supported b y Public Health Service Grants AI15036 and AI07151-06 from the National Institute of Allergy and Infectious Diseases and HL19171 from the National Heart, Lung and Blood Institute. We thank Anne Stephenson for her assistance with these studies, and Lynn Brooks for preparation of the manuscript.
References Barenkamp, S.J., R.S. Munson and D.M. Granoff, 1981, J. Infect. Dis. 143, 668. Batteiger, B., W.J. Newhall V and R.B. Jones, 1982, J. Immunol. Methods 55, 297. Burnette, W.N., 1981, Affal. Biochem. 112, 195. Cannon, J.G., W.J. Black, I. Nachamkin and P.W. Stewart, 1984, Infect. Immun. 43, 994. Hu, P.C., Y. Huang, J.A. Graham and D.E. Gardner, 1981, Biochem. Biophys. Res. Commun. 103, 1963. Johnson, D.A., J.W. Gautsch, J.R. Sportsman and J. Elder, 1984, Gene Anal. Techn. 1, 3. Laemmli, U.K., 1970, Nature (London) 227, 680. Nachamkin, I., J.G. Cannon and R.S. Mittler, 1981, Infect. Immun. 32, 641. Petit~ C., M.E. Sauron and J. Theze, 1982, Ann. Immunol. 133D, 77. Towbin, H. and J. Gordon, 1984, J. Immunol. Methods 72, 313. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350.