Phosphorylation

Sylvain Foisy foisys at medcn.umontreal.ca
Tue Feb 5 11:54:30 EST 2002


Hi,

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!!.

Cordially

Sylvain Foisy, Ph.D.
BIONEQ Manager
Genome-Quebec
Montreal - Quebec
CANADA




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