WB - Transferring REALLY big proteins - (Jun/13/2011 )
I've been trying to detect ATR and ATM by western blot without much success. Aside the possible issues with the antibodies, I'm unsure of what the best transfer conditions are for proteins of this size (300 and 350kDa, respectively). Right now I'm doing it for ~75min but that might be still insufficient for these. Anyone has any experience with this?
what is the composition of the transfer buffer? to transfer large proteins, up to 0.05% sds is recommended (along with 10-20% methanol).
what percentage acrylamide? lower concentrations are recommended for large proteins (5-7%, we've used 3.5-5% gradients to separate very large proteins).
keep in mind that, especially for large proteins, you shouldn't expect complete transfer.
(i cheated and copied my response from another forum)
I would add - what are your transfer conditions (semi-dry, wet, voltage and amperage etc.)?