His-tag fusion protein detection problem - (Feb/20/2006 )

:( Hi all:

I had my His fusion protein (in pDEST 17 vector) expressed in BL21 Star (DE3) cells. The cells grow well (no toxic to cells). But I can't detect protein expression using either anti-His monoclonal Ab (Roche) or polyclonal Ab (Covance). I belive that my plasmid construct is ok. Does anybody has similar problem? How can I detect my protein expression?

Thank you.

Carl

-PCarl-

QUOTE (PCarl @ Feb 20 2006, 07:27 PM)

Hi all:

I had my His fusion protein (in pDEST 17 vector) expressed in BL21 Star (DE3) cells. The cells grow well (no toxic to cells). But I can't detect protein expression using either anti-His monoclonal Ab (Roche) or polyclonal Ab (Covance). I belive that my plasmid construct is ok. Does anybody has similar problem? How can I detect my protein expression?

Thank you.

Carl

You probably don’t have enough protein expressed that is way. You should lowed on the gel supernatant meaning the medium cells were growing in. 2- cell pellet too. Maybe your protein is insoluble. if so add 8 m urea 3x thaw and freeze then centrifuge it again and lode the supernatant as well as the pellet. IT should work

-okice-

I am a little unsure of what you have tried...have you run a gel and done a quantification on your purified sample?

-aimikins-