I would like to monitor cAMP-dependent protein kinase (PKA) activity in differentiated L6 skeletal muscle cell lysate using western blotting. I hope to use the cells treated with Protein Kinase Inhibitor (PKI) as the negative control and would like to monitor the PKA phosphorylation using an antibody on western blot, upon stimulation by an agonist. I hope to use either phospho-PKA substrate or phospo-CREB antibody. Could anyone recommend this or suggest any other alternative of monitoring PKA activity?
You could also do a kinase assay using immuno-precipitated (IP) PKA in the presence of its substrate and a radiolabeled ATP.Then look for the presence of PKA using antiPKA and the presence of phosphorylated PKA substrate using phosphoPKA substrate antibody via western blot analysis. In addition by a western blot you can easily detect the phosphorylated substrate due to the radiolabeled ATP.
I worked on PKB.
URL to thesis:
http://scholar.uwindsor.ca/etd/3367/
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