Special procedures for phosphorylated proteins? - During isolation and western blot (Oct/11/2007 )

I want to check for some phosphorylated proteins in my cells. Normally, I just make an acetone precipitation during RNA isolation and use that for western blotting. Is this also possible for phosphorylated proteins? Or do I have to make cell lysates? Or even isolate proteins in a different way with stabilizers added?
And are there special procedures for the blotting, incubation and developing/staining?

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I do cell lysates using RIPA buffer containing (final concentrations):
50mM Tris-Hcl pH 7.4
1% NP-40
0.25% Na-Deoxycholate
150mM NaCl
1mM EDTA
1mM PMSF
Aprotinin, Leupeptin, Pepstatin 1µg/ml each (protease inhibitors)
1mM Na₃VO⁴ (Don't forget to activate it!)
1mM NaF
NaF, Sodium orthovanadate and NaF are phosphatase inhibitors, which you should certainly use.
And what I do: keep the lysates on ice at all time (except when boiling with loading buffer of course). And always use fresh lysates.
Cheers, JM

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Thanks! Is it still possible to make these lysates from cells that have been (freshly) frozen @-80 for some weeks? Or really use fresh lysates from "fresh" cells?

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The best answer for this is "sometimes." Frozen lysates have worked for me for some things but not others. Fresh lystates are best, but it wouldn't hurt to try frozen lysates in a trial blot.

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