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!
- - - - -

Re-purification of His-tagged protein over IMAC

IMAC His Tag Purification Purity

  • Please log in to reply
2 replies to this topic

#1 Missle



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 166 posts

Posted 21 November 2016 - 11:07 AM

Hello.  Has anyone ever tried taking the elution of a his-tagged protein off of an IMAC column, buffer exchanging back into binding buffer (with the same or higher level of imidazole) and re-purifying over an IMAC column?  I'm wondering if it might be a way to increase the purity by increasing the ratio of tagged protein to host cell contaminants but have never tried it.

#2 mdfenko


    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,383 posts

Posted 22 November 2016 - 04:31 AM

it will work but you may suffer unacceptable losses.

talent does what it can
genius does what it must
i used to do what i got paid to do

#3 labtastic



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 205 posts

Posted 28 November 2016 - 08:43 PM

No, this will do absolutely nothing to improve purity.


Your protein will re-bind...but so will all the other contaminants that bound the first time. If contaminants binds once, they'll bind twice.


This is why expressing proteins with cleavable (solubility) tags (e.g. SUMO, GST, MBP, GFP, etc) and re-running over the column while your un-tagged protein flows through is a wonderful purification step...all the contaminants that bound the first time also bind the second time...while your tag-less protein flows through.


Alternatively, use ion exchange and/or gel filtration if you need higher purity.

Edited by labtastic, 28 November 2016 - 08:46 PM.

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

©1999-2013 Protocol Online, All rights reserved.