Edman sequencing of proteins from 2D gels
1Laboratory of Gene Regulation, Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Japan.
Abstract:
The Western blotting/sequencing technique using polyvinylidene difluoride (PVDF) membrane is one of the most popular technique for Edman sequencing. A protein sample is transferred from a 2D polyacrylamide gel electrophoresis (2D-PAGE) gel onto a PVDF membrane by electroblotting. The membrane carrying the protein is directly subjected to protein sequencing. If sequencing fails after a few cycles, the PVDF membrane is removed from the sequencer and treated with deblocking solution. If this attempt at sequencing fails, alternative methods such as the Cleveland method are required. Because the resolution of 2D-PAGE is high, the combined use of 2D-PAGE, Western blotting, and Edman sequencing often allow effective sequence determination of proteins that could not be easily purified by conventional column chromatography.
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