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

From:"Houston, Ronnie"

Not sure if this will help, but I did some work on antibodies that only
recognized denatured proteins on western blots. 
If I remember correctly, a 5 minute room temperature pretreatment with 1%
sodium dodecyl sulfate (SDS) worked on a couple of antibodies I couldn't get
to work by IHC otherwise. I don't have the actual papers to hand, but the
references are as follows:

Antigen retrieval in cryostat tissue sections and cultured cells by
treatment with sodium dodecyl sulfate (SDS).  		Brown D et al. 
Histochem Cell Biol 1996; 105: 261-267

Improved immunodetection of nuclear antigens after SDS treatment of
formaldehyde-fixed cells.	Wilson DM, Bianchi C
J Histochem Cytochem 1999; 47: 1095-1100

There is another unconventional technique that was suggested some years back
for carbohydrate and protein antigens using N-glycanase F, but I have no
personal experience of the results.

Unconventional antigen retrieval for carbohydrate and protein antigens. Guhl
B et al
Histochem Cell Biol 1998; 110: 603-611

Good luck, let us know how you get on.


Ronnie Houston
Director of Anatomic Pathology
Bon Secours HealthPartners Laboratories
5801 Bremo Road
Richmond, VA 23226
(804) 2877972
(804) 2877906 - fax


-----Original Message-----
From: MacDougall, John []
Sent: Monday, June 21, 2004 11:56 AM
Subject: [Histonet] The use of reducing agents in antigen retrieval?

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.


John MacDougall 

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