2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: application to the analysis of IgG.
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.
Gonzalez
Leticia
L
Department of Integrative Physiology, The University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA.
Bustamante
Juan J
JJ
Barea-Rodriguez
Edwin J
EJ
Martinez
Andrew O
AO
Haro
Luis S
LS
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods* / Q000706:statistics & numerical data; D006041:Goats; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods
D007074:Immunoglobulin G; D021122:Protein Subunits; D012967:Sodium Dodecyl Sulfate