pI and Transfer Buffer - (Dec/17/2008 )
So I normally do my transfers in Towbin buffer +20% MeOH and don't really change that. However, I am about to transfer a protein that my calculations say will have a neutral isoelectric point at pH 8.3 (Towbin pH). I know CAPS buffer has a pH of 11 and my protein will be highly negative according to pI calculations in that buffer.
I just don't want to screw up transfer by using Towbin and not having it migrate, but I have never used CAPS buffer. Anyone have experience with it?
are you transferring from sds-page?
if so, then pI doesn't matter. the sds imparts a net negative charge to the protein.
if too much sds is stripped off the protein by the methanol or if the protein is large then you can add up to 0.05% sds to the buffer.
if you're running native page then you can add 0.05% sds to the buffer.
you can use the caps buffer as well. we used caps when we transferred to pvdf for amino acid sequencing (avoids the glycine).