problem with stable clones - (Nov/30/2004 )

Hi,
I wish to make a stable cell line in HeLa cell expressing my protein of interest (which is also expressed endogenously by the cells). I got several clones after selection with geneticin. But after doing western blot to check the clones, I found that none of them is expressing my protein of interest. I hope somebody will help me with this.

Thanx

Is the endogenously expressed protein being picked up by western blot? If not then it could be your western that's the problem. Also, are you using a positive control?

Hi
The endogenous protein is being picked up by western blot but not the transfected one.

Do you include sodium butyrate in the media 24hours before harvesting? Also, what size of culture flask are you harvesting from?