An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein.
A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required.
Hennessey
J P
JP
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Scarborough
G A
GA
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis; D002417:Cattle; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D008565:Membrane Proteins / Q000032:analysis*; D009113:Muramidase / Q000032:analysis; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010047:Ovalbumin / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013025:Glycine max / Q000201:enzymology; D014361:Trypsin Inhibitors / Q000032:analysis
D008565:Membrane Proteins; D014361:Trypsin Inhibitors; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D009113:Muramidase; D000251:Adenosine Triphosphatases; D002256:Carbonic Anhydrases