Comparison of conditions for immunodetection of antigens using monoclonal antibodies after electrotransfer on nitrocellulose membrane
Villarroya, S.; Scholler, R.
Pathologie-Biologie 33(10): 981-986
1985
ISSN/ISBN: 0369-8114 PMID: 3912709 Document Number: 252930
Solubilized human sperm proteins are separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, electrophoretically blotted onto a nitrocellulose sheet and their antigenicity probed with 5 monoclonal antibodies. Bound proteins are exposed to different nonionic detergents and a comparative study is reported. The use of 0.05% Tween 20 or 0.05% Nonidet P-40 in the blocking and washing buffers and in the immunoprobes causes variations in the patterns of 3 monoclonal antibodies. Washing buffers containing either 0.1% of detergent (Tween 20, Nonidet P-40, Triton X-100) or different concentrations (0.05, 0.1, 0.4%) of Tween 20 remove prebound proteins from the nitrocellulose membrane. Nitrocellulose strips processed with different detergents lack different immunoreactive bands. The 5 monoclonal antibodies recognize degraded antigens and endogeneous proteolysis changes the antibody patterns.