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Specific antiserum staining of two‐dimensional electrophoretic patterns of human plasma proteins immobilized on nitrocellulose
Author(s) -
Anderson N. Leigh,
Nance Sharron L.,
Pearson Terry W.,
Anderson Norman G.
Publication year - 1982
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150030304
Subject(s) - nitrocellulose , glycoprotein , antiserum , sodium dodecyl sulfate , chemistry , chromatography , gel electrophoresis , electrophoresis , blood proteins , resolution (logic) , antigen , collodion , monoclonal antibody , antibody , biochemistry , microbiology and biotechnology , membrane , biology , immunology , artificial intelligence , computer science
Human plasma proteins separated by high‐resolution two‐dimensional electrophoresis have been electrophoretically transferred to sheets of nitrocellulose using a modification of the method of Towbin, Staehelin, and Gordon [8]. Although the proteins have been denatured in sodium dodecyl sulfate and separated into subunits, the nitrocellulose‐bound molecules still react with appropriate specific antisera even after storage of the transfer in air at room temperature for 5 months. Of 25 proteins whose location in the pattern had been previously determined, 24 are specifically revealed on transfers of whole plasma patterns by appropriate antiserum. In addition, 6 previously unidentified proteins (prothrombin, C1s, C4γ, C1s inhibitor, Ig J‐chain, and a 1 AP glycoprotein) have been identified in the pattern for the first time using the transfer technique. It therefore seems likely that a large majority of proteins (> 96 % in this study) retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically. The transfer technique thus constitutes a generally useful immunological “third‐dimension” in the high‐resolution separation of proteins. Of three monoclonal antibodies similarly tested, none could detect antigen transferred to nitrocellulose from a two‐dimensional gel, while each bound specifically to the appropriate antigen absorbed in native form to the nitrocellulose.