Shift in the position of band onto the western blot - (Oct/17/2012 )
A predicted band of 45 kDa appeared in the position of 95 kDa on the western blot . Shift in the position is a very general phenomena for the group of proteins it belongs to. Could you guys help me to explain it? What kind of post-translational modification can be guessed? Please help me.
Denaturing gel or native?
It was on SDS Page. The primary antibody used in this experiment was peptide purified antibody and was used in 1:1000 dilution in 1XPBST (.1% Tween 20). The blot was developed with ECL. The experiment is reproducible.
It's a big big shift; is it a discrete band or a smear?
Are you sure it isn't a cross-reacting band from the antibody (which is a very common phenomenon with antibodies, no matter how they are purified)?
Can you see the un-modified form in the same lane? Do you have a way of reliably making the higher band appear (and what is it)?