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PKA activity assays



Hi!  
     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,
          Melissa Albin 
       
 
 
 

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