[Histonet] HRP conjugation

[Jeff Oliver]

Unfortunately you will need to buy a new primary.  A mouse primary also
may have cross-reactivity problems with mouse samples, so I would stay
away from any preconjugated mouse antibodies too.  Preconjugating your
own antibodies can be a timesaver, but I found it introduced to many
random variables.  The most concerning one in your case is you can't
guarantee all of the secondary is bound to the primary.  I would expect
the same result if you try to preconjugate with an anti-mouse secondary.
-Jeff

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Donin,
Nick (NIH/NCI)
Sent: Friday, November 18, 2005 1:15 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] HRP conjugation

Hello Everyone, 

This isn't a Histology question, but it relates to antibodies, so I
thought someone could help me.  I'm doing a western blot, and I'm
running the protein from mice tumors.  I'm using a mouse primary
antibody to stain for a particular antigen, but the problem is that when
I apply anti-mouse secondary, it stains everything on the blot
(presumably because the tumor tissue is rich in mouse IgG antibodies).
I thought I could get around this by conjugating my primary antibody
directly to H.R.Peroxidase, thus eliminating the need for a secondary.
My first question is:
1. Does this strategy even make sense?
2. Does anyone have a technique or product for carrying out this
conjugation?

 

Thanks for all your help, any thoughts or suggestions are greatly
appreciated. 

 

Nick Donin

CRTA

Neuro-Oncology Branch

National Cancer Institute

National Institutes of Health

9000 Rockville Pike

Building 35, Room 2B-203

Bethesda, MD 20892

 

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet