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
- - - - -

Discoloration of Nickel affinity resins during 6-his protein purification

Nickel affinity 6-his discoloration protein purification

  • Please log in to reply
1 reply to this topic

#1 mjmspano

mjmspano

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 21 October 2013 - 06:33 AM

For the last year I have been plagued with a problem when I purified my 6-his labeled protein-- my resin would become a reddish-brown color that could not be washed away. The color was eluted off when I eluted my protein and I had problems separating it from my protein. I was isolating material from mammalian cell tissue culture.

 

I am an idiot. I was co-purifying transferrin, an iron binding protein that is prevalent in tissue culture supernatant, and will also bind nickel. At some point I stopped loading my material in the presence of 20mM Imidazole. When I re-initiated this practice any color was much less, and the column could be reused upon washing.

 

I would read posts about this problem on this site, which is why I am posting this advice. You must load your starting material in the presence of a low concentration of Imidazole. Especially if you are purifying proteins from tissue culture supernatant.



#2 Hellen

Hellen

    member

  • Active Members
  • Pip
  • 11 posts
1
Neutral

Posted 22 October 2013 - 05:29 PM

Thank you very much. I think this is very usefull for me. I will purify protein from mammalian cell some day.







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

©1999-2013 Protocol Online, All rights reserved.