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.
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.
Castellanos-Serra
L
L
Division of Physical Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba. lila.castellanos@cigb.edu.cu
Vallin
A
A
Proenza
W
W
Le Caer
J P
JP
Rossier
J
J
D000665:Ampholyte Mixtures; D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D007525:Isoelectric Focusing / Q000379:methods; D020033:Protein Isoforms / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D015032:Zinc
D000665:Ampholyte Mixtures; D007093:Imidazoles; D020033:Protein Isoforms; D011506:Proteins; D015032:Zinc