His Tagged Recombinant Protein not Recognized in Western Blot - (May/20/2013 )
I expressed an 18kD protein in e.coli with a 6x His on the N-terminus. It purified fine w/IMAC but I did need to reduce the imidazole in the sample and binding buffer a little from my standard protocol or a lot of it went in the flow thru. I then tried to run a western blot to confirm if an aggregate band was a contaminant or an aggregated form of my protein and used an anti-His tagged antibody that I've used many times before. My monomeric protein isn't really being detected even at ridiculous loading amounts (I titered from 5ug to 50ng) and the positive control is fine. I know there is plenty of protein there and enough histidine residues that it was able to bind to the nickel resin so I'm not understanding why the antibody can't detect it. Any ideas?
I was also thinking of denaturing a sample with a strong denaturant and seeing if that would allow for antibody binding but I don't have much experience with this and wasn't sure if it was a good idea.
Any suggestions or recommended reading would be much appreciated.
The anti-His antibody notoriously can not work.
You'd better change to other tags.