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
- - - - -

Use of carbonate buffer to dilute antibody?


  • Please log in to reply
4 replies to this topic

#1 yo4trad

yo4trad

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 12 October 2010 - 11:55 AM

Is it necessary to use a carbonate type buffer for capture antibody dilution.
Is it ok to use BSA?

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,788 posts
132
Excellent

Posted 12 October 2010 - 12:05 PM

bsa is not a buffer.
talent does what it can
genius does what it must
i do what i get paid to do

#3 teddytariku

teddytariku

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 13 October 2010 - 12:22 AM

Is it necessary to use a carbonate type buffer for capture antibody dilution. Is it ok to use BSA?


The coating buffers most used are 50 mM carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mM phosphate-buffered saline (PBS), pH 7.2 . I don't think you can use BSA to dilute capture antibody.

You may use it to reduce high background when using polyclonal antibodies (PCA)as capture antibody. Polyclonal serum will result in strong signal, and in this case you may use BSA to preabsorb the serum using competitor protein such as BSA. In ElISA, you can use BSA as blocking agent because of its ability to compete with nonspecific factors in the test sample for available plastic sites.

Hope this helps.

#4 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 13 October 2010 - 12:58 AM

I think if you use BSA to coat your plate you'll end up with a plate loaded of BSA and very little antibody.

I've always used either carbonate buffer pH9.6, or PBS to coat ELISA plates. Just dilute you coating antibody directly in this buffer and add to the wells.

#5 sgt4boston

sgt4boston

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 276 posts
1
Neutral

Posted 13 October 2010 - 04:16 AM

All buffer suggestions are good. Carbonate, etc.. do not put BSA in your coating buffer that contains your antibody. You may also 'shock' your antibody by briefly subjecting it to low pH then diluting in your coating buffer and adding to your wells.

Use BSA or other proteins for blocking uncoated plastic surfaces. there are also commercial blockers which you can try. if you wish to keep your plates dry and for extended periods...use some sucrose in buffer as final block step decant and allow plates to dry inverted. then keep dry in plastic bag with dessicant and refrigerate.




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

©1999-2013 Protocol Online, All rights reserved.