PHOSPHOPROTEIN LABELLING - DIFFICULTIES
hgtsei at med-rz.uni-sb.de
Thu Jun 26 11:07:47 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.
> Angus Bell
> abell at tcd.ie
Did you check phosphorylation by SDS-PAGE?
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