That is, an antibody, which binds a protein on a western blot very specifically, appears not to bind at all when the protein has a FLAG or 3xFLAG tag?
Hi Adrian,
As Arjan mentioned, the most possible issue with Flag-tagged proteins is that it might be proteolytically cleaved off and hence not recognized. I had the same problem in the past and I switched to the cell signaling antibody DYKDDDDK. However you could consider changing the tag to a His-tag, which should interfere less with the function of your protein.
Good luck!
First thing to try is to load a positive control expressing the tag to detect the signal. There's something that would be useful to exclude if the tag was proteolytically cleaved off. If the protein is endogenously expressed in your biological model, you should see two bands when incubation with a specific antibody to such protein is performed. The heavier one is the protein fused with the Flag tag.
I'm having the same problem.
https://www.researchgate.net/post/Antibody_for_protein_not_the_tag_does_not_recognize_tagged_protein_Why_could_this_be?cp=re72_x_p2&ch=reg&loginT=4a7cq76kOqY9xFwokBfbwPc2BW7UkeZpEq33sBfKNRLXflyEKKuSww%2A%2A&pli=1#view=53879fd6d11b8be82a8b45c9
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