Large proteins in Western - (Sep/14/2012 )
criscastells on Mon Oct 1 10:09:15 2012 said:
Yes, the standard protein (all of them) are lost during the transference.
Lost from the gel or membrane/blot?
what is the membrane type, pore size?
did you back it with a second membrane?
do you see anything on the membrane? have you stained with ponceau?
sds, if not stripped from the protein during transfer, will interfere with binding. that's why the manual recommends not exceeding 0.05% and adding up to 20% methanol in the transfer buffer.
The standards do not appear in the membrane neither in the gel. Probably I use too much SDS. I know it is not possible to transfer completely all the proteins but my problem is that the big ones (from approximatelly 100KDa) remain in the gel using normal conditions.
I will try again using 20%MeOH ans 0,01%SDS. I think I will also increase transfer time.