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In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis
Author(s) -
Ploug Michael,
Stoffer Bjarne,
Jensen Arne L.
Publication year - 1992
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.1150130130
Subject(s) - electroblotting , membrane , chemistry , chromatography , cysteine , alkylation , polyvinylidene fluoride , in situ , organic chemistry , biochemistry , nitrocellulose , enzyme , catalysis
For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (> 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and in situ alkylation after electrotransfer onto polyvinylidene difluoride membranes. Precautions against NH 2 ‐terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.