I overexpressed Flag-tagged FH (fumarase) by transfected pcDNA plasmid in 293T cells. After 3 days, I harvested the cell and extracted the whole protein. Then, I performed WB to test transfection efficiency.

The results showed that when I use the antibody against FH, I can only see endogenous FH, but not Flag-tagged FH which has a bigger molecular weight. However, When I use the antibody against Flag, I can detect the overexpressed Flag-FH.

The data, from my view, means the FH primary antibody cannot recognize the exogenous expressed Flag-tagged FH protein. Why? Will the tag impair the primary recognition? But the epitope of the FH antibody is G354, the Flag tag locates at N-terminal.