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!
- - - - -

How to distinguis soluble from insoluble recombinant proteins produced in E. col

  • Please log in to reply
1 reply to this topic

#1 csalat



  • Members
  • Pip
  • 2 posts

Posted 02 July 2012 - 01:01 AM

Hii! I am gonna transform E.coli with a plasmid to express the protein and I need to know whether it is soluble or insoluble. I know that I should open the cells, separate soluble and insoluble fractions by centrifigation and load each on the SDS polyacrylamide gel. But I would truly appreciate if anyone could give me an effective protocol to do that.


#2 Papaver



  • Active Members
  • PipPipPipPipPip
  • 77 posts

Posted 02 July 2012 - 11:27 AM

My suggestion (the way I had done before): Grow your cells in 25 -50 ml LB culture and induce the protein expression for 2-4 hours at a OD600 = 0.6. Then take a 1.5 ml to 2 ml sample and pellet the cells. Resuspend the cells in lysis buffer (e.g. 200 µl): protein buffer + lysozyme (check out for concentration, it's been a long time when I did it so I can't remember) + Dnase. Incubate for 30 min on ice. Then freeze-thaw the cells (freeze in liquid nitrogen, thaw at room temperature) and repeat this 3 - 5 times. Then spin for ~ 30 min at 4°C, and transfer the supernatant in a new eppi. Then you can resuspend the pellet = insoluble fraction in SDS loading buffer and dilute the supernatant = soluble fraction with loading buffer. Run a SDS-Gel. It depends on the amount of protein if SDS-PAGE is enough or if you need to do a western blot.
I hope I haven't forgotten any step. It worked fine for me and I could start with the 1 L culture...

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

©1999-2013 Protocol Online, All rights reserved.