Hello Soham,

Getting a good transfer efficiency with large proteins might take a little bit more time. It can be hard to move big molecules around quickly. So, for 250kDa protein use 25-30V overnight (16 hours) in a 4-degree cold room.

You should also consider other factors which I have listed below, if you have missed out.

1. Choosing the right gel is a key factor in the successful transfer of high molecular weight proteins. Proteins >200 kDa are compacted into a very narrow region at the top of the running portion of the gel, leading to poor resolution of protein bands. By using Tris-Acetate gels which maintain a pH of around 7, high molecular weight proteins can migrate further through the gel, allowing increased distance between protein bands thereby separating out large molecular weight proteins with higher resolution. This will allow better transfer of high molecular weight proteins out of the gel leading to increased transfer efficiencies and higher sensitivity.

Large proteins will move out of a lower percentage gel much more efficiently than out of a higher percentage gel, and they will also resolve better. So, you may use a low percentage gel such as 7%.

2. The composition of your transfer buffer is critical. Large proteins can precipitate out in the presence of methanol. Avoid this by decreasing the methanol percentage (10% or less) in your transfer buffer. Larger proteins can precipitate in the gel, inhibiting their transfer. The SDS added during SDS-PAGE usually takes care of this problem, but larger proteins might require some more SDS. So, you may add up to 0.1% SDS in your transfer buffer to discourage precipitation. However, SDS can inhibit binding of proteins to membranes. So, try starting with a lower concentration of SDS (for instance, 0.0375%).

3. For large molecular weight proteins use PVDF membrane for transfer. Large proteins can precipitate out in the presence of methanol. PVDF membranes do not require any methanol in the transfer buffer. So, you have a higher chance of successfully transferring your protein to the blot using these membranes. Just don’t forget to activate the membrane with methanol before using it.

4. Electrophoretic transfer can be done in either semi-dry or wet conditions. Wet conditions are usually more reliable as it is less likely to dry out the gel, and is preferred for larger proteins.

Good Luck!