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
- - - - -

Blocking small molecules


  • Please log in to reply
1 reply to this topic

#1 skylarhjk

skylarhjk

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 23 September 2009 - 07:54 AM

I am having trouble with non-specific binding of a small biotinylated molecule as part of a competitive ELISA. I have found that the small molecule binds non-specifically to a variety of blocking agents. I have tried serum protein based blockers as well as a protein-free blocking buffer. In each case, the small molecule is detected more strongly in a blocked well than in a well not blocked at all. This suggests that the blocking agents are interacting with the conjugated molecule and are counter-effective. Below is the molecule that I cannot seem to block, that I am using to bind to coating antibody and detecting with HRP-streptavidin:

5-methoxytryptamino/PEG4/biotin

Any insight would be greatly appreciated.

#2 sgt4boston

sgt4boston

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
1
Neutral

Posted 24 September 2009 - 10:53 AM

In a "non-blocked at all well" your SA-HRP conjugate does not bind to the naked plastic surface?

There are also fish based blockers and peptide based materials if your background is truly blocker analyte related.

Do you have no background issue with ab coated, blocked plate and hrp conjugate or with blocked plate with conjugate only?

I wonder if instead of using the biotinylated molecule to compete with the analyte you used an HRP conjugated molecule?




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

©1999-2013 Protocol Online, All rights reserved.