[Histonet] RE: antigen retrieval/enzyme digestion

[Todd Sherman]

On Sun, 08 May 2005 12:00:26 -0500,  
<histonet-request <@t> lists.utsouthwestern.edu> wrote:

> Today's Topics:
>
>    1. RE: antigen retrieval/enzyme digestion (Patsy Ruegg)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sun, 8 May 2005 09:42:06 -0600
> From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
> Subject: RE: [Histonet] antigen retrieval/enzyme digestion
> To: "'Till, Renee'" <TillRenee <@t> uams.edu>,
> 	<histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <200505081542.j48FgR75001351 <@t> pro12.abac.com>
> Content-Type: text/plain;	charset="us-ascii"
>
> I have heard of it being done both ways depending on the epitope of
> interest.  In general I do HIER first and then EIER.
> Patsy
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Till,  
> Renee
> Sent: Monday, May 02, 2005 5:55 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] antigen retrieval/enzyme digestion
>
> When  trying both on the same slides, which would go first, the antigen
> retrieval or the enzyme digestion?
>
>
> Renee' Till, HT
> Arkansas Childrens Nutrition Center
>
> 1120 Marshall
>
> Little Rock, AR
>
> 72202
>
> (501) 364-2774

Renee,

I'd tend to agree with Patsy as far as a sequence best determined  
empirically. You'll probably need to compare each protocol for each  
antigen of interest.

 From a theoretical perspective, both techniques of "unmasking" involve the  
breaking of strong molecular bonds. HIER is typically designed to cleave  
the basic side-chains of amino acids on proteins previously fixed (or  
bound to each other) with x-aldehyde. Enzyme digestion is similarly  
designed to cleave proteins but at well defined amino acids which are not  
necessarily basic - it depends on the digesting enzyme and its substrate.

My guess would be that progressing from a more basic (read simple/generic)  
to a more specific cleaving sequence would be more efficient;  
consequently, treat with HIER first and enzyme digestion second. The HIER  
would open up the tissue to further access and the enzyme digestion could  
then more specifically cleave regions hindering an even more localized  
access to your target antigen. To a great extent, these success derived  
 from these sequences will be greatly influenced by the local milieu of  
your target antigen. The relative content of various amino acids in a  
given milieu may or may not be known. To that end empirical observations  
are "best" which is why mandating one particular protocol over another may  
be a bit dubious.

Good luck.
Todd


President
HistoSoft Corporation
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