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
- - - - -

High background using 5% BSA for blocking Western Blot


  • Please log in to reply
3 replies to this topic

#1 schaapje_86

schaapje_86

    member

  • Active Members
  • Pip
  • 12 posts
1
Neutral

Posted 09 December 2010 - 04:27 AM

I compared blocking my nitrocellulose membrane with 1% BSA and 5% BSA. I found that a higher percentage of BSA caused a higher background signal on my membrane. Do you have any idea why that it?Will 5% skimmed milk will be better as a blocking agent?
Thanks a lot!

#2 knuf

knuf

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 104 posts
3
Neutral

Posted 09 December 2010 - 06:24 AM

In general, I prefer 4% milk in TBST over 5% BSA unless there is some reason I have to use BSA (such as for phospho-epitope antibodies). If I do need to use BSA, I block overnight instead of for the 1-2 hours that I block with milk.

#3 BioMiha

BioMiha

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 225 posts
8
Neutral

Posted 09 December 2010 - 11:10 AM

In my experience as well, milk is a better blocking agent than BSA. We also observe that some antibodies generally give a high background. Did you observe this in different experiments or just that one?

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,242 posts
336
Excellent

Posted 09 December 2010 - 11:34 AM

Milk is also no good for alkaline phospatase conjugated antibodies as the milk contains phosphatases leading to increasing your background. Milk is fine for HRP though.




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

©1999-2013 Protocol Online, All rights reserved.