Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel.
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.
Hirano
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Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki, Japan.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007887:Fabaceae / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis; D015151:Immunoblotting; D008832:Microchemistry; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis*; D010946:Plants, Medicinal; D012639:Seeds / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis
D000596:Amino Acids; D006023:Glycoproteins; D010936:Plant Extracts; D010940:Plant Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean