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.
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.
Ploug
M
M
Institute of Biochemical Genetics, University of Copenhagen, Denmark.
Stoffer
B
B
Jensen
A L
AL
D000478:Alkylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003545:Cysteine; D004586:Electrophoresis / Q000379:methods; D005069:Evaluation Studies as Topic; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D011145:Polyvinyls; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D003545:Cysteine; D008565:Membrane Proteins; D008567:Membranes, Artificial; D009113:Muramidase; D011145:Polyvinyls; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; C524898:PLAUR protein, human; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers