Puzzled

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Sat Oct 13 12:49:20 EST 2007


On Thu, 11 Oct 2007, Yvonne Couch wrote:

> I have what sadly may be assumed to be a silly question..if I am
> stimulating my cells with an unknown substance and I want to know what
> cell surface receptor it is binding to how do I go about this without
> systematically knocking down every known receptor or running a
> microarray.

An 'unknown substance' sounds quite exciting. Can you make it radioactive?
If it came from space or something, it may already be; if not, collecting
it from cells fed radiolabelled nutrients or iodinating it in vitro might
do it. Then, you can put it on your cells, crosslink the surface with
something (glutaraldehyde, maybe even something more vicious like osmium
tetroxide?), lyse, run a gel, blot or dry down, and do an autoradiogram.
You'll have to use lysis and electrophoresis conditions which won't break
the crosslink. You'll hopefully have a band where your
receptor-crosslinked-to-unknown-substance is. This gives you an idea of
its molecular mass, at least; if you can run a 2D gel, you get the pI as
well.

You could have a go at cutting the band or spot and sending it for mass
spec, but there are likely to be too many overlapping bands or spots in
the area to get a straight answer, especially given that most cell surface
molecules are in rather low abundance. You could try doing a plasma
membrane prep after crosslinking and before running the gel, which would
dramatically simplify the mixture and so might enable mass spec. I think
there are various ways to do this, but i did it once by gentle lysis and
ultracentrifugation, which was pretty simple. The bright side is that if
you do get an MS hit, it will simultaneously tell you the receptor and the
ligand!

> Is it possible?

You're a scientist: anything is possible. Given enough time and funding :).

If you don't get a good answer here, wander over to the biochemistry
department and talk to Iain Campbell; he probably won't be able to give
you an answer either, but he can make it a question in one of the absurdly
difficult data handling exercises he gives the undergraduates, and maybe
one of them'll come up with something!

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk



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