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
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
----- 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,
> 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
> 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
> step to what is already an overnight incubation.
> Histonet mailing list
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