![]() ![]() The size of the target protein should be considered when choosing transfer conditions. In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into a cassette along with protective fiber pads. Electrophoretic transfer can be accomplished under wet or semi-dry conditions. The transfer buffer used for electroblotting is similar to gel running buffer with the addition of methanol which helps proteins bind to the blot. The gel and membrane are assembled into a sandwich along with several sheets of filter paper which protect the gel and membrane and help to ensure close contact between their surfaces. Taking PVDF membrane for example, the PVDF membrane is placed between the gel and the positive electrode so that the negatively charged proteins migrate from the gel onto the PVDF membrane. In general, PVDF is much better for low molecular weight (MW) proteins. ![]() Compared to NC membrane, PVDF membranes offer better protein retention, physical strength, and chemical compatibility. There are two common membrane types used for western blot (WB) analysis, polyvinylidene difluoride (PVDF) and nitrocellulose (NC). Transfer is performed by passing a current across the gel to the membrane. Due to the speed and efficiency of transfer, electroblotting is the method relied upon in most laboratories. Protein Transfer from Gel to Membrane in Western Blot Assayįollowing gel electrophoresis, the separated protein mixtures are transferred to a solid support for further analysis. ![]()
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