Shift in the position of band onto the western blot (View forum version)



chimera

Posted 16 October 2012 - 10:13 PM

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.

bob1

Posted 16 October 2012 - 11:33 PM

Homework?

Denaturing gel or native?

Antibody conditions?

chimera

Posted 17 October 2012 - 01:29 AM

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.

bob1

Posted 17 October 2012 - 12:39 PM

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)?