Re: [Histonet] Biotin free detection of goat primary antibodies

From:"Gayle Callis"

The avidin/biotin blocking step does not take that much time, 15 minutes for 
each step and is done BEFORE you apply the primary antibody for overnight 
staining.  It will also depend if your tissue (you did not say what you are 
staining, kidney, liver, brain???) is one that contains a higher level of 
endogenous biotin.

Omitting the primary antibody is not a good negative control, you should be 
running a Goat IgG control, at the same concentration as your primary 
antibody.  It could be that biotin is not the cause of your background 
problem but nonspecific binding of the Goat IgG of your primary antibody to 
other tissue components.

Biocare has a single link polymer kit that is specific for goat but you 
still need to run a correct negative control, goat IgG. This can be 
purchased from Jackson Immunoresearch and is not expensive.

Jackson now has bovine secondaries, which are supposed to reduce background 
when working with large animal primary antibodies.  Look into this also.

Gayle M. Callis
Bozeman MT

----- Original Message ----- 
From: "Min-Han Tan" 
Sent: Monday, May 26, 2008 8:55 PM
Subject: [Histonet] Biotin free detection of goat primary antibodies

> Dear all,
> I would like to enquire - I am using a goat primary antibody currently, 
> and
> my negative control (omitting primary antibody) is showing background
> staining - this is likely a result of biotin.
> I'd like to enquire if any one is familiar with biotin free systems that 
> can
> detect goat antibodies. A search of the Dako / Envision product line does
> not yield any results. I am very reluctant to add an avidin-biotin 
> blocking
> step to what is already an overnight incubation.
> Thanks!
> -- 
> /min-han
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