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The immunodetection of brain proteins blotted onto nitrocellulose from fixed and stained two‐dimensional polyacrylamide gels
Author(s) -
Jackson Peter,
Thompson R. J.
Publication year - 1984
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.1150050107
Subject(s) - nitrocellulose , coomassie brilliant blue , horseradish peroxidase , chromatography , chemistry , staining , polyacrylamide , stain , peroxidase , polyacrylamide gel electrophoresis , spots , antiserum , biochemistry , biology , antibody , enzyme , membrane , polymer chemistry , genetics , immunology
Proteins visualised by Coomassie Blue staining of two‐dimensional polyacrylamide gels of complex mixtures of brain proteins were blotted onto nitrocellulose sheets. The Coomassie Blue stain was transferred simultaneously and bound strongly to the nitrocellulose, giving a copy of the original gel pattern. Using specific antisera and a second antibody coupled to horseradish peroxidase, proteins in the original mixture could be detected on the nitrocellulose in quantities less than 25 ng. The Coomassie Blue and horseradish peroxidase stains were distinguishable by a suitable filter, allowing precise correlation of spots stained by Coomassie Blue with spots stained with the brown immunoperoxidase reaction product. The technique was used to identify related proteins in electrophoretograms of brain extracts from different animals.