Hi Sebastian,

I did not add a linker between the Flag and the start of my protein.

When I performed transient transfection in 293T cells, both the Flag antibody and the protein antibody worked in WB. Also the Flag IF also worked. It seemed everything was good in 293T cells.

Actually, when I added the HiBiT-tag to the C-terminal, WB showed several bands slightly lower than the expected molecular weight, I suspected the protein might be cleaved and the cleaving site was very near the N-terminal.

Now I plan to do the Flag IF in stable cell line to see whether the Flag is really there. According to your suggestion, transiently transfected cells and checked the FLAG antibody staining.

Thanks for your answer.

Best regards,

Jun