[Histonet] Re: The use of reducing agents in antigen retrieval?

[Todd Sherman]

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John,

It is quite common, and in my limited experience almost expected, that
many pAb and mAb that work under the denatured conditions of a western
blot fail miserably for the IHC protocol. Now we never tried IHC antigen
retrieval under specific reducing conditions other than to use a more
acidic buffer. You can try different buffers with acidic, basic, or
neutral pH to optimize the protocol for your specific Ab-antigen
interaction, but I'm unaware of adding DTT or 2-mercaptoethanol to the
retrieval buffer to achieve desired results. Maybe some other readers have.

An assumption on my part is that generic and wholesale elevated protein
reduction will wreak havoc on your tissue since all proteins will be
exposed equally under "reducing agent" retrieval. Sounds like a nice
empirical study to me. I hope you'll post your results. :)

Hope this helps,
Todd


Todd Sherman
President
HistoSoft Corporation
"Biology in a new form..."
Home: www.histosoft.com
Member Services: www.myhistosoft.com

histonet-request <@t> lists.utsouthwestern.edu wrote:
|
| Today's Topics:
|   10. The use of reducing agents in antigen retrieval?
|       (MacDougall, John)
|
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|
| Message: 10
| Date: Mon, 21 Jun 2004 11:55:35 -0400
| From: "MacDougall, John" <JMacDougall <@t> CuraGen.com>
| Subject: [Histonet] The use of reducing agents in antigen retrieval?
| To: <histonet <@t> lists.utsouthwestern.edu>
| Message-ID:
|     <AE1DCA7140C6334D9D550A1E2D404C77177FB8 <@t> ENTERPRISE.CURAGEN.COM>
| Content-Type: text/plain;     charset="iso-8859-1"
|
| Dear Histonet
|
| We have been trying (unsuccessfully) to use a polyclonal antibody
raised against a peptide in some IHC runs.  The antibody recognizes
antigen by western blotting only under reducing conditions (i.e. DTT or
2-mercaptoethanol).  Thus, it appears that the peptide/epitope is masked
by disulphide bond(s).  Is there an antigen retrieval method out there
that incorporates reducing conditions?  Any help will be appreciated.
|
| Thanks
|
| John MacDougall
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