Hello Rajdeep Roy

In addition to using 6% resolving gel, thickness of the gel is an important factor to consider. Large molecular weight proteins transfer more effectively from thinner gels than from thicker varieties. There is less distance for proteins to migrate during electrophoretic transfer in a 1mm gel than in a 1.5mm gel which may require longer transfer and high molecular weight proteins may get transferred less efficiently.

Always use wet tank transfer to detect high molecular weight proteins. The composition of your transfer buffer is also important. Large proteins can precipitate out in the presence of methanol. You may avoid this by decreasing the percentage of methanol (use 10% or less) in your transfer buffer. Additionally, just to ensure that the protein does not precipitate out, you may add SDS to a final concentration of 0.1%. SDS adds uniform negative charge to proteins, making it easier for them to transfer from the gel onto the membrane.

For high molecular weight proteins, use PVDF membrane (0.45um pore size) for transfer because large proteins can precipitate out in the presence of methanol, and PVDF membrane does not require any methanol in the transfer buffer. Since large molecular weight proteins will transfer out of the gel slowly, I recommend transferring overnight (around 16 hours) at 4°C at 20-30V instead of using 4 to 6 hours for transfer.

Best.