Hi everyone,

I generate a stable cancer cell line with Flag-tagged protein, Flag is in N-terminal. The sequence is fully correct.

While the band is very strong on WB using an AB against the protein and the size is also correct, but there is no signal when I blot for Flag. The positive control (transient transfection of this plasmid in 293T) is fine, so the antibody works. Then I performe IF using Flag AB in this stable cancer cell line, the signal is very obvious.

Then I move the Flag to the C-terminal, I still have this problem. It works in 293T cell line. In stable cancer cell lines, AB against the protein works on WB and IF of Flag also works. But there is still no band for Flag on WB.

This protein predominantly in nucleus.

So anyone can help to figure this out? How can I detect the fused protein with anti-Flag antibody?

Thanks!