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
- - - - -

Can I improve my primary antibody?

serum primary antibody purify recombinant protein

  • Please log in to reply
2 replies to this topic

#1 kmobri08

kmobri08

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 28 May 2012 - 06:03 PM

I am trying to create an antibody using some recombinant protein. We received the recombinant protein from another lab and sent it out to be made in rabbits. We received the d70 bleed from the company and tested it immediately by running a gel with the recombinant protein, protein from a WT strain, and protein from a deletion strain. We detect 1 band on the Western for the lane with the recombinant protein, but all other lanes have multiple bands and it is difficult to say if we are detecting our protein of interest in those lanes.

We would like to clean up the antibody so that we don't see all of these other bands. Is there a way that we could do this by utlizing protein from our deletion strain? Or can we only do this with affinity purification using our recombinant protein? My boss mentioned that we may be able to take advantage of the fact that we have a deletion strain, but I'm not sure how we would do this? I did a quick Google search, but all of my results were about affinity purification wiht the antigen. Do I have any other options? It appears that this antibody has promise based on its detection of the recombinant protein, but at this point it is useless if we try to detect the protein in total cell lysate because the signal from other bands is too strong compared to what we think is our band in the lysate.

Any help will be very much appreciated! Thanks!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,516 posts
371
Excellent

Posted 28 May 2012 - 07:06 PM

Did you try titrating the amount of serum used to detect the protein on a blot. I have a rabbit serum against my protein that I use in 1:2000, where I get a single band, but if I use it at 1:1000 or smaller dilution, I get multiple bands.

Is the antibody against native protein? If so, it may not recognise the denatured protein on a western blot very well.

Affinity purification is the way to go for getting specific antibody out of the serum, this way you only get the IgG for the antibody you want, without getting all the other IgG species that are in there as well.

Edited by bob1, 28 May 2012 - 07:07 PM.


#3 klinmed

klinmed

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
1
Neutral

Posted 29 May 2012 - 12:13 PM

I agree that titrating your antiserum may help, as could immunoaffinity purification. A simple first step may be to use lysates of your deletion cells to try and absorb out any contaminating reactivities. A helpful reference:


Giovanni Covini, Edward K.L. Chan, Massimo Colombo, E.M. Tan, Comparison of protocols for depleting anti-E. coli antibody in immunoblotting of recombinant antigens, Journal of Immunological Methods, Volume 190, Issue 1, 28 March 1996, Pages 143-145, ISSN 0022-1759, 10.1016/0022-1759(95)00288-X.
(http://www.sciencedi...02217599500288X)
Abstract: A common problem in immunoblotting for the detection of specific antibodies to recombinant proteins synthesized in E. coli is the presence of contaminating antibodies to E. coli proteins. Four protocols for the efficient depletion of anti-E. coli antibodies from human sera are provided and their uses are discussed.
Keywords: Anti-<span style='font-style: italic'>E. coli</span> antibody depletion; Autoantibody; Immunoblotting

Hope this helps





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

©1999-2013 Protocol Online, All rights reserved.