ISSN:
0173-0835
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 electro-blotted 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.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501401134
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