PHOSPHOPROTEIN LABELLING - DIFFICULTIES

[Thomas Seib]

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.
> 
> Thanks,
> 
> Angus Bell
> abell at tcd.ie

Did you check phosphorylation by SDS-PAGE?

Matthias

-- 
Thomas Seib
Kantstr. 12
66292 Riegelsberg