Conditions suitable for immunochemistry typically try to reproduce the
conditions in which the antibody-antigen binding reaction would
normally/naturally occur, and that is the physiologically isotonic
environment in which it might occur.
Hence, the conditions require buffering near neutrality (pH 7.0 to 7.5),
which is usually done with phosphate, unless the illumination step (enzyme
reaction is affected by phosphate presence in some way, in which case Tris
[pKa=8.1] is used). Sodium chloride is added from 0.1 to 0.15 M; at 0.15 M
it would be about isotonic in a 10-20 mM buffer salt. That is, PBS or TBS
is exactly what you and most people use.
Borate is probably not a good buffer to use. First, borate's pKa is a bit
on the high side and its buffering capacity at neutrality would be suspect,
and second, borate binds to the vicinal diols present in
oligosaccharide/sugar moieties, and if your antigen is a glycoprotein to
which your antibody reacts, you could be affecting the binding.
When in doubt, follow (the consensus of) the literature.
<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. Surely, the closer to physiological conditions the
better. 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.
>>> Thanks
>>> Rubic
>>>>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0
