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In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes: cleavage at tryptophan residues with 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine to obtain internal amino acid sequence.

Analytical Biochemistry

metabolism, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Cattle, Chickens, Electrophoresis, Polyacrylamide Gel, Glass, Humans, Immunoblotting, Indoles, Membranes, Artificial, Molecular Sequence Data, Polyvinyls, Protein Binding, Proteins, Skatole, analogs & derivatives, Tryptophan

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      Proteins immobilized to glass-fiber supports and polyvinylidenedifluoride membranes are cleaved in situ with a tryptophan residue-specific reagent, 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. Protein fragments can be eluted from polyvinylidenedifluoride membranes after in situ digestion and electrophoresed and electroblotted to new polyvinylidenedifluoride membranes for subsequent sequence determination of selected bands. Five proteins (bovine serum albumin, ovalbumin, beta-casein, beta-lactoglobulin, and myoglobin) of known sequence containing one to three tryptophan residues each were used as model substrates to evaluate the specificity and extent of cleavage. Under the conditions used, the reaction is rapid and exhibits a high degree of specificity for tryptophan residues since N-terminal sequence analysis of the digested, immobilized samples yields the expected, newly generated N-termini. No detectable cleavage occurred at nontryptophan residues nor at acid labile aspartic acid-proline peptide bonds. Solution cleavage reactions were also performed and the resulting digest was analyzed by gel electrophoresis. We found a positive correlation between the solution and in situ cleavage reactions, in that, proteins which show cleavage in the solution reaction also yield internal sequence data after in situ digestion. Since tryptophan occurs with low frequency in proteins, this cleavage reaction has the potential to generate a small number of fragments and in favorable circumstances allow sequence analysis of the unfractionated reaction mixture. In addition, the sequence obtainable after, but not before, in situ digestion can be used as an indication that the N-terminus of the protein is blocked.

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