Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Abstract

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.