Antibody buffer: Why does my recipe have such high osmo?

D.K. dk at
Fri Oct 11 08:47:00 EST 2002

tmorris at wrote:
>Are there any experts on the practical aspects of immunocyt? If so I thank you
> in advance for advice on this one.
>Our lab has a hand-me-down recipe for antibody buffer, for staining imobilized
> isolated neurones for fluorescent microscopy. It's basically a tris based
> buffer which includes 500mM NaCl. No one can offer a rational explanation for
> this, nor can I find anything similar on the web or in books. I've used a
> borate buffer with a more normal osmolarity (300 mOsmol) and the staining was
> just as good. I suspect PBS would work also, which is what most books
> reccomend. 
>Such high salt might neutralize static charges on IgG, but why would this help
> in binding epitope. Am I missing somthing? The person who designed this buffer
> must have had a good reason and I don't want to just ditch a protocol without
> a clear understanding of the whys and wherefores.

If these are fixed permeabilized cells, high salt buffer is not necessary
but can frequently be helpful to decrease level of non-specific staining 
quite dramatically (you do control staining with different cells and different
antibody, right?). 

If these are live cells, it make absolutely no sense. 


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