So you think that protein transfer for western blotting is a piece of cake? Consider these important tips before proceeding:
- Use only high-quality, analytical grade methanol. Impure methanol can increase transfer buffer conductivity and yield a poor transfer.
- In many cases, ethanol can be substituted for methanol in the transfer buffer with minimal impact on transfer efficiency. Check this using your samples.
- Do not reuse transfer buffer since the buffer will likely lose its ability to maintain a stable pH during transfer.
- Do not dilute transfer buffers below their recommended levels since this decreases their buffering capacity.
- Do not adjust the pH of transfer buffers unless specifically indicated. Adjusting the pH of transfer buffers can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
- Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases binding of the protein to nitrocellulose membrane. PVDF membrane can be substituted for nitrocellulose when SDS is used in the transfer buffer.
- Addition of SDS increases the relative current, power, and heating during transfer, and may also affect antigenicity of some proteins.
- Increasing methanol in the transfer buffer decreases protein transfer from the gel and increases binding of the protein to nitrocellulose membrane.