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
- - - - -

HELP! Western Blot: Anti His detection problem


  • Please log in to reply
1 reply to this topic

#1 Leen

Leen

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 20 May 2010 - 07:22 AM

Hi everybody!

To detect a His tagged protein on a WB I'm using a His-Tag monoclonal antibody from Novagen which is supposed to recognize 5 consecutives histidine residues regardless of the surrounding amino acid context. My protein has a 6His tag on the N-term position but it is not detected and I dont understand why. I used several positive controls with a Nterm His tag and again, no detection. However, a protein with a Cterm His tag was detected in my WB. But my antibody is supposed to have no Hi tag position specificity.....Do you have any possible explanations for that? (Im working on denaturing conditions so I dont think my His tag is stucked in the protein...).
Thanks in advance for your help.
Leen

#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 20 May 2010 - 08:58 AM

We had problems with detection of his-tagged proteins on western blot using his-tag specific antibodies from several different vendors. I wrote to GenScript and asked them if they would send me a sample of their his-tag-specific monoclonal antibody, and it worked wonderfully, even against proteins expressed from pET16b, which attaches a 10x his-tag (GenScript's site only mentions their antibody's use with 6x, 5x, and 4x his-tags, so I asked them to let me try it out on a 10x-his-tagged protein before buying it, and they agreed).




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

©1999-2013 Protocol Online, All rights reserved.