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 precipitated after buffer exchange


  • Please log in to reply
1 reply to this topic

#1 netnus

netnus

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 31 posts
0
Neutral

Posted 30 January 2005 - 11:46 AM

Dear friends,

I have been facing this trouble for a while: I want to exchange the buffer for my protein. I use Amicon Centriplus YM-3 with MWCO=3,000. My protein is soluble and stable in buffer A: KH2PO4 75mM,NaCl 75mM,4mM 2-Mercaptoethanol,2mM EDTA and 1mM DTT,5mM NaN3). The new Buffer B is:0.5M imidazole.HCl,NaEDTA 2mM and DTT(1mol/mol protein). After exchange for 4 times, the solution is concentrated down to 1ml. Then I store it in the -20 degree freezer. I thaw it and spin it down . My protein is almost completely precipitated.
Had anyone ever met with such a problem? How did you solve it?Any suggestion will be greatly appreciated!


netnus

#2 snolan6

snolan6

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 02 February 2005 - 12:18 AM

Have you made sure the pH of your buffer is permissive to your protein being soluble? Bad stuff like that happens to me sometimes when I try out a new protocol or make up a new buffer recipe, and pH is always my first candidate cause.

Also, make sure your pH electrode is functioning properly - ours has given me problems a few times.

Edit - the temp of your buffer can affect the pH also....

Edited by snolan6, 02 February 2005 - 12:28 AM.





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

©1999-2013 Protocol Online, All rights reserved.