I have a human protein that is tagged at the N-terminus with a 1x flag tag followed by an HA tag. After transient transfection of this vector into 293Ts and protein harvest, I can detect the protein by western blot with an anti-HA antibody but not the anti-flag antibody (Sigma Flag M2). Has anyone had issues with a 1x flag tag? I know the anti-flag antibody works because I can detect another protein that is flag tagged.
Kozak - 1xFlag - HA - ORF
Any idea what is going on here?