3 replies to this topic

[sara.r]

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

[BioMiha]

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).

[sara.r]

BioMiha, on 05 November 2010 - 02:14 AM, said:

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).

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.

[BioMiha]

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?