I want to evaluate the Gi protein signalling events to measure the desensitisation of receptor signalling. Instead of GTPgammaS is there any other method for evaluating the desensitisation of signalling by Gi.
This is not my precise field so forgive me if i miss the target a little but i believe a classic system is to co express GIRK (the guanine nucleotide binding (G) protein-activated Potassium channel) with your receptor of interest record the Potassium currents from the GIRK channel in response to receptor activation. You can do this in mammalian cells or Xenopus oocytes. Oocytes and Two electrode voltage clamp is less taxing than mammalian cell patch clamping, especially if you have no electrophysiology background.An example review would be Life Sci. 2001 Apr 27;68(22-23):2525-33.but there are many more recent papers out there if you search Pubmed for GIRK or Gi signaling.
You can also try the cAMP EIA kit from Amersham or Cayman.This assay is based on the competition between free cAMP and a cAMP tracer for a limited number of cAMP-specific antibody binding sites. Because the concentration of the cAMP tracer is held constant while the concentration of cAMP varies, the amount of cAMP tracer that is able to bind to the rabbit antibody will be inversely proportional to the concentration of cAMP in the well.
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