An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: Its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis
- 30 May 2001
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 22 (9) , 1677-1685
- https://doi.org/10.1002/1522-2683(200105)22:9<1677::aid-elps1677>3.0.co;2-h
Abstract
A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA‐IEF) or immobilized pH gradient (IPG) gels by in‐gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole‐Sodium dodecyl sulfate (SDS)‐zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry‐compatible silver staining, but simpler and faster. Proteins were digested in imidazole‐SDS‐zinc stained CA‐IEF and IPG gels in the presence of a zinc‐chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high‐sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.Keywords
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