Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.
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.
Feldman
Galia
G
Ethrog (Invitrogen Israel) Ltd., Ness Ziona, Israel.
Bogoev
Roumen
R
Shevirov
Julia
J
Sartiel
Adam
A
Margalit
Ilana
I
D000906:Antibodies / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*
D000906:Antibodies; D006639:Histidine; D019793:Fluorescein