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Author Topic: Antigenic peptide competition in IHC  (Read 4141 times)

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Offline AB

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Antigenic peptide competition in IHC
« on: August 09, 2006, 05:27:15 PM »
Hi,

I am trying to perform antigenic peptide competitions by pre-incubating my primary antibody with the antigen (1:50 ratio) overnight, pre-clearing prior to staining. The staining is higher in the competition slides when compared to primary antibody alone. No background due to secondary. Can someone tell me why? If you have exerienced this, do you have a protocol or technique that worked?

Would really appreciate your help.

AB

Antigenic peptide competition in IHC
« on: August 09, 2006, 05:27:15 PM »

Offline Rollypoke

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Re: Antigenic peptide competition in IHC
« Reply #1 on: September 04, 2009, 12:38:51 PM »
If it makes you feel any better, I'm having the same problem. I've only JUST begun to trouble-shoot it, so i don't quite have a solution yet. If you find one, let me know, and I'll do the same!

Cheers

Offline richard03

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Re: Antigenic peptide competition in IHC
« Reply #2 on: September 05, 2009, 03:49:58 PM »
Did you dilute the primary antibody after overnight incubation? Be sure the final concentration of competitive primary antibody is same as your non-competitive primary antibody.

It is best to perform peptide competition control at concentrated form rather than diluted form. 1:50 may be to much. I would use 1:5 or 1:10 in concentrated form and then dilute it to appropriate concentration afterwards.



Re: Antigenic peptide competition in IHC
« Reply #2 on: September 05, 2009, 03:49:58 PM »