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Selective proteome‐wide detection of hydrophobic integral membrane proteins using a novel fluorescence‐based staining technology
Author(s) -
Hart Courtenay,
Schulenberg Birte,
Patton Wayne F.
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200406001
Subject(s) - integral membrane protein , transmembrane protein , proteome , membrane protein , staining , chemistry , bacteriorhodopsin , fluorescence , stain , sodium dodecyl sulfate , gel electrophoresis , biophysics , membrane , biochemistry , biology , physics , receptor , quantum mechanics , genetics
Integral proteins containing two or more α‐helical membrane‐spanning domains are underrepresented in two‐dimensional gels. While sodium dodecyl sulfate (SDS)‐polyacrylamide gels separate these proteins, staining profiles are usually dominated by high‐abundance hydrophilic proteins in the specimen. A fluorescence‐based stain is presented that selectively highlights integral proteins containing two or more α‐helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5–10 ng of bacteriorhodopsin, a seven‐helix transmembrane protein. Stained proteins are detected using a laser scanner or charge‐coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F 1 F 0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.