PHOSPHOPROTEIN LABELLING - DIFFICULTIES

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Wed Jun 25 10:10:35 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.

So, you are saying it's not enzyme, nor substrate, nor ATP. Then I can see 
only one possibility: G-50 chromatography for some reason goes wrong
(no separation of protein from ATP or all protein sticks to your Sephadex).
Is the person doing this the same person as before? 

- Dima




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