loooking for protein phosphorylation method
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Mon Feb 18 15:26:41 EST 2008
Am 12.02.2008, 04:28 Uhr, schrieb prabhat khadka <mebewafa from yahoo.com>:
> If anyone could help me with protein phosphorylation protocal , taht
> will be highly appericated.
> hope to see soon
If you provided a little more info on what you want to do it would be
easier to help. So looking into my crystal ball I'd guess you want to
metabolically label proteins with 32P (or 33P, which I personally prefer
unless double- or triple labelling is required).
In that case you incubate the cells in medium containing the isotope in
the form of phosphate, with as much label as local regulation allows you
to use in a single reaction. There should be no other source of P in the
medium, but you need glucose to get that phosphate into ATP. After
incubation (time and conditions depend on the experiment) spin, wash and
homogenise the cells (for the small volume involved a ball-bearing
homogeniser is nice, but a Potter will also do).
The next step involves isolation of the compartment you want to
investigate, usually by fractionated centrifugation. Any compartment other
than cytosol would be "opend" in then. Soluble proteins are separated from
small molecules (which contain most of the radioactivity) by gel
filtration on a small, disposable column. Membrane proteins are separated
by centrifugation instead.
Next you separate the proteins, e.g. by 2D-electrophoresis followed by
autoradiography. If you can do that with a phosphor-imager you already
have quantitative results, otherwise you have to cut out the spots of
interest, digest the gel and count the radioactivity in a beta-counter.
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