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

From:"C.M. van der Loos"

Dear Min-Han,We solved this problem with a 3-step approach: goat primary - rabbit anti-goat IgG - anti-rabbit polymer/HRP. The second step we purchased from Southern Biotech Associates, Birmingham, AL. Dilution of the rabbit anti-goat IgG (human IgG adsorbed) came out at 1:2000-5000 (30 min, RT). Any anti-rabbit polymer/HRP will do. Gayle is fully right with respect to the negative control: find a non-immune goat IgG with a known IgG protein concentration. You need that to calculate the final dilution.Hope this helps a bit.Cheers, ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The NetherlandsDate: Tue, 27 May 2008 10:55:59 +0800
From: "Min-Han Tan" 
Subject: [Histonet] Biotin free detection of goat primary antibodies
To: histonet@lists.utsouthwestern.edu

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.


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