PHOSPHOPROTEIN LABELLING - DIFFICULTIES
FAVE at nitfix.edu.ar
Thu Jun 26 23:14:05 EST 1997
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?
<fave at nitfix.edu.ar>
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