How do I optimize transfer conditions to avoid poor transfer problems for my western blot experiment?

One way is to increase the current or transfer time to encourage the movement of larger proteins. However, one should do so carefully as this may lead to issues such as overheating and affect the transfer of smaller proteins. Alternatively, one can optimize the transfer buffer by adding a small amount of sodium dodecyl sulfate (SDS) and reducing the amount of methanol. This adjustment enhances the solubility of larger protein molecules, thereby improving their migration during the transfer process. Additionally, make sure to pre-wet the membrane with a polar organic solvent like methanol or ethanol (for PVDF membranes). One should also saturate the filter paper and blotting pads thoroughly with a transfer buffer, making sure to eliminate any bubbles during assembly. If the gel is excessively flattened, remove some pads to ensure that the blotter can be closed without applying excessive pressure.