Western blotting/Edman sequencing using PVDF membrane.

Abstract

The western blotting/Edman sequencing technique using polyvinylidene difluoride (PVDF) membrane is one of the most popular technique for determination of primary structure. A protein sample is transferred from a SDS-polyacrylamide gel electrophoresis (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 two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is high, the combined use of 2D-PAGE, western blotting, and Edman sequencing often allows effective sequence determination of crude proteins mixture that could not be easily purified by conventional column chromatography.