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PKA activity assays
I am a new subscriber, a Masters student of University of Toronto
and I am working on a project looking at PKA activity in EBV transformed
B cells (we are looking at the enzyme activity in Bipolar Disorder).
I have been working on optimizing the PKA activity assay using
Kemptide as a substrate. I am currently working on optimizing the
assay and I am trying to use the PKA inhibitor PKI(5-22) to
determine the PKA activity in the cytosolic and membrane fractions of
the cells (by counting PKA activity as that which is inhibitible by
PKI). I have been using a concentration of 5uM for PKI, protein
concentrations ranging from 3ug to 20 ug, Kemptide at 200uM,
Mg(75mM)/ATP(100uM) and cAMP at 25uM. I will eventually want to do a
dose-response with respect to cAMP for the final study. With these
parameters the PKI has been inhibiting approximately 35% of total kinase
activity, although this has yet to be a consistent result, and has only
been achieved in the cytosolic fractions.
I would really appreciate if anyone could shed some light on any
of these issues for me. If anyone is working on PKA activity in crude
cell extracts,I would be very interested in any information you might be
able to share with regards to the particular controls that you are using
to ensure that the activity being measured is PKA specific.
Thank you very much,
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