Analysis of Glucose-6-Phosphate Dehydrogenaseafter Purification usingGel Electrophoresis and Western Blotting
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The matrix used in our work was a polyacrylamide gel of 5% concentration of the acrylamide monomer crosslinked to N,N'-methylene bisacrylamide (bisacrylamide). In order to estimate purity and molecular weight in electrophoresis the surfactant sodium dodecyl sulfate (SDS) is used. b-mercaptoethanol (BME) was applied to reduce the disulfide bonds and allow determination of migration by molecular weight only and not by shape. The proteins in the gel can be visualized by staining the gel with silver or Coomassie Brilliant Blue R-250 after electrophoresis. The next step in our protein analysis is to do a Western Blot, which is a more specific test that allows us to visualize antibodies bound directly to G6PD. Separated proteins in a gel can be transferred to a nitrocellulose membrane for Western Blot analysis. All sites on the membrane which do not contain blotted protein from the gel can then be non-specifically "blocked" so that antibody will not non-specifically bind to them causing a false positive result.To detect the antigen (G6PD) blotted on the membrane, a primary antibody specifically directed against G6PD is added at an appropriate dilution and incubated with the membrane. In order to detect the primary antibody which has bound, an anti-immunoglobulin secondary antibody coupled to a reporter group such as fluorescein is added (e.g. Goat anti-rabbit IgG H&L, Fluorescein) resulting in a visible band where the primary antibody bound to the protein that can be seen under ultraviolet light.