Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

no expression of my protein


  • Please log in to reply
1 reply to this topic

#1 rosepolaris

rosepolaris

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 06 February 2009 - 01:04 PM

I was trying to test the expression of a dimer (AB), one (A) with Histag, the other (:D without. I also created a disulfide bond between A and B to stabilize the dimer.

The cloning was very successful. A and B are on different vectors, hense different antibiotics. I co-reansformed them into BL21(DE3) STAR.
I picked up one colony and grew in 5ml LB at 37C overnight. Then I inoculated 1ml O/N culture into 200ml LB, added IPTG to 1mM when the OD600 reached 0.6. Expression was at 22C O/N.

The next day, I did purification under denaturing condition (since I have already tried under native condition and got nothing). I didn't elute the proteins and just ran the beads. However I did not see bands for my proteins on SDSPAGE. I also ran pre-induction sample, lysate, flow-thought sample, and didn't see right-sized bands for my proteins.


What are potential pitfalls in protein expression? or, maybe I just picked up a wrong colony.....

#2 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 12 February 2009 - 10:22 PM

Hey

Do you see any expression when you transform them independently?

The problem is expression or coexpression?

TC




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.