2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: Application to the analysis of IgG
| Autor: | Andrew O. Martinez, Luis S. Haro, Edwin J. Barea-Rodriguez, Leticia Gonzalez, Juan J. Bustamante |
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| Rok vydání: | 2006 |
| Předmět: |
Chromatography
Staining and Labeling Goats Protein subunit Clinical Biochemistry Permanganate Sodium Dodecyl Sulfate chemistry.chemical_element Zinc Sensitivity and Specificity Biochemistry Oligomer Stain Analytical Chemistry Staining Silver stain Protein Subunits chemistry.chemical_compound chemistry Immunoglobulin G Animals Electrophoresis Gel Two-Dimensional Protein Structure Quaternary Polyacrylamide gel electrophoresis |
| Zdroj: | ELECTROPHORESIS. 27:2016-2023 |
| ISSN: | 1522-2683 0173-0835 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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