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Native immunoblotting: Transfer of membrane proteins in the presence of non‐ionic detergent
Author(s) -
Bjerrum Ole J.,
Selmer Johan C.,
Lihme Allan
Publication year - 1987
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.1150080905
Subject(s) - electroblotting , nitrocellulose , chemistry , agarose , chromatography , gel electrophoresis , sodium dodecyl sulfate , membrane , electroelution , covalent bond , membrane protein , electrophoresis , biochemistry , polyacrylamide gel electrophoresis , enzyme , organic chemistry
For analysis with monoclonal antibodies against discontinuous epitopes and for performance of functional characterization it is important to maintain proteins native during electrophoresis and electroblotting. However, in vitro membrane proteins only remain soluble and native in the presence of non‐ionic detergent, which, on the other hand blocks the subsequent protein immobilization on nitrocellulose. With model proteins from human erythrocytes, platelets and‐serum, it has been examined if electroblotting to nitrocellulose can be performed from agarose gels in the presence of non‐ionic detergent (native blotting). Each of the following procedures were successful: (i) employment of detergents with low critical micellar concentration; e. g. Berol EMU‐043 (C 16–18 , E 10 ); (ii) insertion of a 2–3 mm thick detergent‐free agarose layer between the gel and nitrocellulose; (iii) transfer at pH 11.8 of immunoprecipitated proteins after gel wash; (iv) binding via covalent coupling to vinylsulfone activated nitrocellulose. The procedure to be chosen depends on the type of analysis and on the required binding efficiency. Compared to immunoblotting after sodium dodecyl sulfate‐gel electrophoresis, native immunoblotting was 10–30 times more sensitive. Of general applicability is the easy transfer from crossed immunoelectrophoresis gels and the possibility for performance of covalent coupling to nitrocellulose during transfer with preservation of the original binding properties of the matrix.