anti his antibody for western blotting - high background in cell lysate western blotting (Nov/03/2010 )

Hi
I am doing some western blotting for an intracellular his tagged recombinant protein in CHO cell lysate, I tried anti his HRP from roche and anti penta his from qiagen followed by conjugated second antibody. with anti his HRP I didnt get any band even in 200 microgram lysate but giagen antibody in its minimum titer (1/1000) has high background. does any body has any suggestion for an anti his antibody with high sensitivity and specifity in crude cell lysate?

regards

We've done some stuff with an anti-his mAb from Sigma. It seems to work OK. My question is what sort of conjugated secondary are you using (specificity, company).

BioMiha on Fri Nov 5 10:14:25 2010 said:

I used goat anti fab antibody (affinity purified from anti fab poly clonal antibody)from sigma, my coworkers are using 1/3000 titer for purified samples I tried 1/3000, 1/6000, 1/10000 titers and I always had a lot of non specific bands.

I am not sure why you chose to use an anti-Fab secondary antibody. What source is the recombinant protein that you're trying to express? If you don't see anything with the anti-his from Roche, you might have problems with the expression level. Be sure to run a WB with a positive control, so you'll at least know if the anti-his antibodies work. Are the anti-his monoclonal or polyclonal?