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

[tmorris at uhnres.utoronto.ca]

Yes these are fixed, permeablized cells, so the the reduced background explanation is probably it. I'll play with it and see if this makes a difference to my preps /antibodies, although non-spec staining isn't usually a problem.

Thanks,

Rubic



D.K. wrote:

> tmorris at uhnres.utoronto.ca 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. 

> DK





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