Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.

Autor: Feldman G; Ethrog (Invitrogen Israel) Ltd., Ness Ziona, Israel., Bogoev R, Shevirov J, Sartiel A, Margalit I
Jazyk: angličtina
Zdroj: Electrophoresis [Electrophoresis] 2004 Aug; Vol. 25 (15), pp. 2447-51.
DOI: 10.1002/elps.200405996
Abstrakt: The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band.
(Copyright 2004 Wiley-VCH Verlag GmbH and Co.)
Databáze: MEDLINE
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