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
- - - - -

protein refolding


  • Please log in to reply
1 reply to this topic

#1 chn09

chn09

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 42 posts
1
Neutral

Posted 26 August 2010 - 09:03 PM

Hello everyone.
Im trying to purify alkaline phosphatase enzyme that has been overexpressed in E. coli BL21DE3. The protein formed inclusion bodies upon IPTG induction. I was able to isolate and purify the protein in the inclusion bodies using denaturing Nickel affinity chromatography. Then i used dialysis to remove the denaturant (8 M urea)

1. After i dialysed the unfolded protein for 48 hrs in a cold room. next day i was able to see a white precipitate at the bottom of the dialysis bag. I assumed it is the salt, but my colleague told it was the refolded protein itself.
2. I performed the assay after concentrating the dialysed samples using a speed vac. Still that white precipitate was visible.However the assay worked.

Was that white precipitate a salt or refolded protein?. Is dialysis not enough to remove urea?
pls help

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,787 posts
132
Excellent

Posted 26 August 2010 - 10:41 PM

it was most likely your protein, but not all of it (that's why your assay worked). you may avoid this precipitation if you reduce urea stepwise and slowly.
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.