His-tag protein expression - (Jul/16/2010 )
I have tried to expression the Hect domain of Drosophila Nedd4 by cloning the domain into pQE30. I have tried to expression the construct, which is in frame, in BL21 E.coli at 30C, IPTG induced, for 3hrs but could not get anything. I also loaded the pellet and the lysate after sonication and did a western blot with anti-His antibodies but did not get any signal.
Is there any suggestion?
What level of IPTG are you using to induce. I have used 0.4mM and seems to work well when I induce for 1.5hrs at 30degrees Celsus. After lysis, have you boiled (95C) your pellet in loading buffer and loaded it directly on your SDS gel.