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2‐D native‐PAGE/SDS‐PAGE visualization of an oligomer's subunits: Application to the analysis of IgG
Author(s) -
Gonzalez Leticia,
Bustamante Juan J.,
BareaRodriguez Edwin J.,
Martinez Andrew O.,
Haro Luis S.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500634
Subject(s) - staining , coomassie brilliant blue , silver stain , chemistry , permanganate , chromatography , gel electrophoresis , polyacrylamide gel electrophoresis , stain , protein subunit , negative stain , biochemistry , microbiology and biotechnology , biology , electron microscope , inorganic chemistry , genetics , physics , gene , optics , enzyme
A 2‐D native‐PAGE/SDS‐PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver‐staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie‐staining (R‐250, G‐250), metal ion‐reverse‐staining (zinc, copper), and fluorescent chromophore‐staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS‐PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R‐250/permanganate silver‐staining combination detected IgG subunits using 2 pmol of sample. Coomassie G‐250 and native fast silver staining in the first‐dimensional gel produced detectable subunits in the second‐dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first‐dimensional gel did not produce discernible subunits in the second‐dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2‐D native‐PAGE/SDS‐PAGE system, Coomassie staining of the first‐dimensional native gel combined with permanganate silver staining of the second‐dimensional denaturing gel provides the most sensitive method (2–3 pmol) for visualizing constituent subunits from their oligomeric assemblies.