foisys at medcn.umontreal.ca
Tue Feb 5 11:54:30 EST 2002
In article <3C56A4D8.EE5D2753 at ix.urz.uni-heidelberg.de>,
Leo Serrano <mz6 at ix.urz.uni-heidelberg.de> wrote:
> Hi Everybody,
> I am looking for a protocol to label in vivo a protein, using
> phosphate. I do understand this could be extremely nasty and lousy
> experiment, but I am looking for details about this. I already establish
> a IP protocol that works perfectly for my condictions, and I am just an
> easy way to determine whether my protein is phosphorylated... any
> suggestion are welcome..!!!
I did many such experiments while studying phosphorylation of
cytoskeletal proteins. Ok, here goes:
1- Prepare a phosphate-free medium that is the closest to the one you
are using regularly. Add HEPES to buffer since you might have to
incubate your cells out of the cell culture incubator. I my case, I
incubated my cells in a bacteria incubator without the CO2, thus the
HEPES. In addition, labelle phosphate comes in the form of phosphoric
acid, with the expected effect on pH...
2- Get the labelled phosphate with the highest specific activity
possible. I worked exclusively with 2000Ci/mmol phosphate coming from
Amersham. This allow you to add minimal volume to your labelling mix.
3- Use between 0.25 to 1 mCi/ml of labelled PO4 per plate and minimal
volume of medium. I incubated my cells in 10 cm plates with 4 ml of
medium. Incubate for 4 hours at 37C in a plexiglass box inside the
incubator, if possible also having a plexiglass door.
4- After the incubation, get the plates out, aspirate the medium and
discard it in the liquid bottle (with the usual dusty stuff). Washed
carefully twice the cell layers with ice-cold PBS and put on plastic
tupperware-like boxes contaning ice (already installed in the
radioactive manipulation hood) as level as possible; add minimal amount
of lysis buffer and keep it there for 30 min.
5- Proceed with your IP protocol. Be careful of the radioactive wastes.
The beads are very hot even after thorough washes: the labelled PO4
stays there but will be eliminated in the SDS-PAGE process.
Dispose of your wastes ASAP and be very careful about touching anything
with potentialy contaminated gloved hands.
If you need any additionnal infos, feel free to ask me. I have a written
protocol, but it is in french!!.
Sylvain Foisy, Ph.D.
Montreal - Quebec
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