Hello everyone,
I am working on Adenylyl cyclase (AC) cell lines overexpressed in HEK. For WB, I always get good bands with AC antibodies but with respect to Flag-tag antibody, I have not received any bands. I have tried different things, using fresh buffers, TBS, TBST, different concentration of Flag and anti-mouse HRP (secondary antibody) but no bands are coming. My colleague uses the same antibody but she gets results always.
I use 10, 20 and 40 ug of my proteins but still challenging for this. I appreciate if anybody could help me in this matter.
Sincerely,
If you are able to observe the AC overexpression in the anti-AC immunoblot, it seems like this might be an issue with the Flag tag on your AC protein, especially since the antibody works for your colleague. I'd verify that the (1) you are using the same tag as your colleague and (2) that the tag is actually present at the DNA level.
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