PHOSPHOPROTEIN LABELLING - DIFFICULTIES

[Gabriel Favelukes]

In article <33B11291.5882 at mail.tcd.ie>, ABell at tcd.ie wrote:
#We have been labelling proteins with [32P] ATP for protein
#serine/threonine phosphatase assays, and have encountered difficulties
#of late.  The method used is basically that described by Mackintosh
#(1993), Protein Phosphorylation: a Practical Approach pp 197-230. 
#Briefly, the dephosphorylated protein is incubated with labelled ATP and
#a protein ser/thr kinase in an appropriate buffer, and then the reaction
#products are separated on Sephadex G-50 and the phosphoprotein peak
#(detected by scintillation counting) collected.
#
#This used to work beautifully for us but now we get no labelled
#phosphoprotein peak, only ATP.  We have tried three different suppliers
#of cAMP-dependent protein kinase catalytic subunit, another kinase,
#casein and phosphorylase kinase as substrates, different buffers, and
#different labelled ATP stocks, all to no avail.
#
#Does anyone out there who does this have any idea what might suddenly be
#going wrong?  All suggestions are welcomed, even if they seem stupid! 
#We just cannot figure out what has changed.

Have you checked if there is any 32P-labelled protein formed at all, e.g. 
by precipitating and washing with cold 5% TCA?

Luck!
Gabriel Favelukes
<fave at nitfix.edu.ar>