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. |
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Authors: | M Ploug B Stoffer A L Jensen |
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Affiliation: | Institute of Biochemical Genetics, University of Copenhagen, Denmark. |
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Abstract: | 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 (greater than 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 electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-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. |
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