Following gel electrophoresis, the separated protein mixtures are transferred to a solid support for further analysis. Due to the speed and efficiency of transfer, electroblotting is the method relied upon in most laboratories. Transfer is performed by passing a current across the gel to the membrane. There are two common membrane types used for western blot (WB) analysis, polyvinylidene difluoride (PVDF) and nitrocellulose (NC). Compared to NC membrane, PVDF membranes offer better protein retention, physical strength, and chemical compatibility. In general, PVDF is much better for low molecular weight (MW) proteins.
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. The transfer buffer used for electroblotting is similar to gel running buffer with the addition of methanol which helps proteins bind to the blot. Electrophoretic transfer can be accomplished under wet or semi-dry conditions. In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into a cassette along with protective fiber pads.
Size of The Target Protein
The size of the target protein should be considered when choosing transfer conditions. Smaller proteins will transfer out of the gel faster and may actually transfer through the PVDF membrane into the filter papers beyond. PVDF with a smaller pore size can be used for small proteins and peptides. If there is a suspicion that the protein is transferring through the PVDF membrane, then a second membrane can be included behind the first to catch proteins that migrate through. In contrast, large proteins can be slower to elute and may be retained within the gel, so overnight wet transfer is usually preferred.
Common Transfer Buffer Recipes
|Wet Transfer||Semi-dry Transfer|
|Recommended for smaller proteins, especially proteins smaller than 30 kDa.||Generally faster, better suited for larger proteins greater than 100 kDa.|
|Tips: Transfer time and voltage may require optimization. Over-transferring can occur and thus caution must be taken, especially for small proteins.|
Protein Transfer Protocol
Note: Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.
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