Denaturing protein - protein won't fall apart (Jan/11/2005 )
I am working with a protein that has a theortical size of 17kDa. It has 3 isoforms that are theoretically capable of binding together. There are no cyseine residues in the protein but it has a leucine zipper. We presume the isoforms bind together through the zipper.
We are trying to do Westerns and I have treated the sample with SDS/beta-mercaptoethanol. The protein size on the gel is approximately 46kDa but we can never see the 17kDa fraction.
Potentially I have 2 problems: 1) an antibody that does not work such that it is not specific; 2) an antibody that is correct but I cannot get the protein to fall apart.
Would you have any suggestions for alternatives on denaturing proteins?
Which transfer membrane did you use for your western blot?
Some membranes are specially designed for small proteins (<20 kDa).
I also find the blotting condition is very crucial too!
Does your protein present in abundance?
If not, you might want to try a more sensitive detection system (e.g. ECL-Plus instead of ECL).
Hope this helps.
Thank you for the reply eleceyes. I use a PVDF membrane for blotting. I did wonder if that was a problem but I can see the ladder at 14.3 kDa. So I am not sure. Theoretically, if I can see 14.3kDa then I should be able to see a 17kDa protein as well.
The protein is not that abundant.
I am now wondering about glycosylation or other sort of mechanism that will make the protein larger than predicted and not allow it to fall apart.