[Histonet] HRP conjugation

[Adam Perry]

What do you mean by "It stains everything on the blot"?  Are you detecting bands across the entire MW spectrum?  If so, that sounds more like your antibody concentrations are too high.  I would think if it really were cross-reaction with endogenous mouse IgG that you would detect bands corresponding to heavy and light chain IgG, etc. and not pick up everything in the blot?  I am fairly new to western blotting, but maybe other folks could weigh in with their thoughts...
 
 Adam
 
 Department of Physiology and Biophysics
 University of Illinois at Chicago
 Chicago, IL 60612

"Donin, Nick (NIH/NCI)" <doninn <@t> mail.nih.gov> wrote: 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



		
---------------------------------
 Yahoo! FareChase - Search multiple travel sites in one click.