Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by western blotting.
A technique has been developed for efficient deblocking and subsequent microsequencing of N-terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electroblotted proteins with N-terminal acetylserine or acetylthreonine could be deblocked on-membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas-phase protein sequencer. Similarly, N-formylated proteins could be deblocked on-membrane in HC1 solution and then directly sequenced from the N-terminal amino acid. Proteins with N-terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N-acetylated proteins were also enzymatically deblocked with acylamino acid-releasing enzyme (AARE) after on-membrane digestion with trypsin to generate the N-terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N-terminal sequence analysis of N-blocked protein immobilized on PVDF membrane.[1]References
- Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by western blotting. Hirano, H., Komatsu, S., Kajiwara, H., Takagi, Y., Tsunasawa, S. Electrophoresis (1993) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg
