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
- - - - -

DTT in GST elution buffer


  • Please log in to reply
1 reply to this topic

#1 mcb56

mcb56

    member

  • Active Members
  • Pip
  • 29 posts
0
Neutral

Posted 27 July 2010 - 11:40 AM

Hello, I am purifying proteins via a GST tag. My elution buffer, which works well, is 10mM Tris, 150 mM NaCl, 1mM EDTA, 1mM DTT, 1% Trition, and 20mM Glutathione. I would like to know if it is ok to eliminate the DTT from my elution buffer (and my wash buffer) and still get binding of the fusion to the glutathione column. I should also note that the only way I can get my protein to bind is if I dilute my crude extract 1:5 (perhaps because of excess glutathione in the suspension). I want to eliminate the DTT because it affects my BCA protein assay.

Thank you

#2 bsksln

bsksln

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 27 July 2010 - 06:56 PM

That does not matter much.
DTT is used to created a reducing environment, thus prevent the formation of unwanted bisulfate bond.
You can eliminate DTT in your buffer and run a SDS-PAGE to see if there is a dimer. Some protein do form bisulfate bond and you can see a band above your protein(twice the size of your protein). Do not boiling before loading because it may break the bisulfate bond.
if not, you can proceed your next expriment.




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

©1999-2013 Protocol Online, All rights reserved.