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

[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

Ronnie Houston
Director of Anatomic Pathology
Bon Secours HealthPartners Laboratories
5801 Bremo Road
Richmond, VA 23226
(804) 2877972
(804) 2877906 - fax
ronnie_houston <@t> bshsi.com

  


-----Original Message-----
From: MacDougall, John [mailto:JMacDougall <@t> CuraGen.com]
Sent: Monday, June 21, 2004 11:56 AM
To: histonet <@t> lists.utsouthwestern.edu
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.

Thanks

John MacDougall 







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