loooking for protein phosphorylation method

Dr Engelbert Buxbaum via methods%40net.bio.net (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>:

> hi
> 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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