using unstained protein ladder for western blot

It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency.We recommend pre-equilibrating the gel in 2x Transfer buffer (without methanol) containing 0.02–0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x transfer buffer containing 10% methanol and 0.01%SDS.

For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. This inhibition is higher for nitrocellulose than for PVDF. SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes.

Increase voltage, current or length of time for transfer.

Using unstained protein ladder for western blot