in-gel kinase assay

DREWES at MPASMB.DESY.DE DREWES at MPASMB.DESY.DE
Tue Feb 21 20:10:54 EST 1995


4Fergus Mckenzie <Mckenzie at naxos.unice.fr> wrote:

>Hi!,		 I'm having problems with poor reconstitution of activity in
>in-gel Kinase assays to dose MAP kinase activity. I'm performing whole cell
>lysis followed by either direct migration on the gel or else
>immunoprecipitation of MAP kinase , then loading the gel . Has anyone performed
>an in-depth analysis of critical parameters, detergents to avoid, ATP
>concentrations etc? Thanks for any help possible.
>								
>						Fergus R.McKenzie


For a time, we have performed in-gel-assays of protein kinase activity
routinely. Our procedure was modified after the orginal Geahlen et al. paper
and has been published (Neuron 11, 1993, 153-163). The following points may be
helpful:
i) Use 0.5 mm thick gels for faster renaturation and washing. 
ii) dilute gamma 32P-ATP with cold ATP (try 10 to 100 micromolar) to avoid
nonspecific absorption 
iii)try Mn and/or Mg 
iiii)include PKA catalyt. subunit as a control (commercially available
i.e. Promega). This kinase works very reproducibly in in-gel assays and
allows you to estimate activities of unknown samples
Actually, we have tried with MAP kinase and it worked well under our
conditions.

Hope this helps,

Gerard


From: drewes at crystal.desy.de
Gerard Drewes
Max-Planck-Society
Structural Molecular Biology Unit
DESY, Notkestrasse 85, 22603 Hamburg
Germany




More information about the Methods mailing list