Deblocking and Subsequent Microsequence Analysis of N-Terminally Blocked Proteins Immobilized on PVDF Membrane

Abstract

In recent protein microsequence analyses, proteins separated by polyacrylamide gel electrophoresis (PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane can be easily sequenced by a gas-phase sequencer (Matsudaira, 1987). However, even if the proteins are successfully separated by PAGE and electroblotted onto PVDF membrane, the N-terminal amino acid sequences of proteins with a blocking group at the N-terminus cannot be determined by Edman degradation. A large number of proteins are known to be N-terminally blocked (Aitken, 1990; Tsunasawa and Sakiyama, 1992). For example, 51 of 72 seed proteins in the rice and 14 of 36 seed proteins in the winged bean which were separated by two-dimensional gel electrophoresis were estimated to be N-terminally blocked (Hirano, 1989; Komatsu et al., 1992). It is, therefore, important to establish simple and rapid deblocking and sequencing techniques for these proteins.