How to determine the Epac activity?

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Pablo Andrés Muñoz Llancao

For EPAC activity you have to do a pulldown assay for RAP1. The assay will tell you how much Rap1-GTP (active Rap) is in your sample which is link to EPACT activity. You will have to include the positive control with EPAC agonist 8-pCPT.

For PKA activity , western blot with the phospho-substrate antibody from cell signaling and phosphoCREB or an Eliza assay.

Bruce Allen

There are kits available for assaying PKA activity.

Edward Humphries

As Pablo mentioned most people use the active-Rap1 assay for Epac1 activation. With additional antibodies this can used for other small GTPases that bind to GST-RalGDS-RBD. You can either make this protein as I did in my recent publication or purchase the kit from various places such as CST or Thermo. Rap1 is also activated by PKA and therefore I would use the super Epac agonist 8-pCPT-AM to asses if Epac is involved in you signalling pathway. Feel free to message me if you want any more information as this is very close to what I just completed my PhD on.