Selective proteome-wide detection of hydrophobic integral membrane proteins using a novel fluorescence-based staining technology.
Integral proteins containing two or more alpha-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.
Proteomics Section, Molecular Probes, Inc., Eugene, OR 97402, USA. courtenay.hart@probes.com
Courtenay
C
Hart
Birte
B
Schulenberg
Wayne F
WF
Patton
D001436:Bacteriorhodopsins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D020543:Proteome
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D020543:Proteome; D001436:Bacteriorhodopsins