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
- - - - -

Immunofluorescence: simultaneous polyclonal and monoclonal staining to same prot


  • Please log in to reply
1 reply to this topic

#1 crom80

crom80

    member

  • Active Members
  • Pip
  • 23 posts
1
Neutral

Posted 03 May 2013 - 05:35 AM

Hi,

I am looking at the localization of Protein X during UV irradiation.
I noticed that increased fluorescence of protein X using a monoclonal antibody within 30 minutes of UV irradiation. Since the increase is in a very short time period I do not think it is due to increased expression or decreased degredation (if so, that itself would be a lone paper).
I am hypothesizing that since I used a monoclonal antibody, UV irradiation caused a shift in protein folding or uncovering of the antigen binding site, hence the increase in immunofluorescence signal.
So I am planing to stain with a polyclonal antibody.

I was wondering, if I simultaneously incubate the samples with mouse polyclonal and rabbit monoclonal to the same protein, would I be too optimistic in thinking I would see good staining? Should I just do the two antibodies separately?
And if I go ahead with simultaneous staining, should incubate the two primary antibodies at the same time or one after the other? (monoclonol first or polyclonol first?)
I tried to search the literature for any suggestions but I could not find any attempts such as this.

Thank you.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,246 posts
336
Excellent

Posted 04 May 2013 - 01:11 AM

Should work fine, just watch out for compatibility of the secondaries.




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

©1999-2013 Protocol Online, All rights reserved.