Journal
ELECTROPHORESIS
Volume 25, Issue 15, Pages 2486-2493Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/elps.200406001
Keywords
fluorescence; alpha-helices; integral membrane proteins; p Phi roteomics
Funding
- NCI NIH HHS [R33 CA093292] Funding Source: Medline
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Integral proteins containing two or more a-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 alpha-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 F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.
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