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Gαs agonist assay
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| Basal cAMP falls out of the lowest measurable limit in the linear part of the cAMP standard curve (i.e., cAMP level too low) | Insufficient number of cells | Increase the number of cells in the assay reaction |
| Basal cAMP exceeds the highest measurable limit in the linear part of the cAMP standard curve (i.e., cAMP level too high) | Too many cells | Decrease the number of cells in the assay reaction |
| Agonist-stimulated cAMP level undetectable (i.e., cAMP level too low) | cAMP rapidly degraded by endogenous phosphodiesterase | Include phosphodiesterase inhibitors such as IBMX |
| Cells not expressing sufficient quantities of receptor | Select for higher expressing clones as determined by qPCR or by FACS |
| Compounds not efficacious | Test other compounds known to be efficacious in modulating cAMP production |
| Insufficient compound binding to the receptor | Allow for longer incubation of the assay reactions |
| Agonist-stimulated cAMP level exceeds the linear part of the cAMP standard curve (i.e., cAMP level too high) | Phosphodiesterase inhibition too strong | Test the presence and absence of phosphodiesterase inhibitor |
| cAMP detection antibody too sensitive | Select for an anti-cAMP antibody with lower affinity for cAMP |
| Too many cells | Decrease the number of cells in the assay reaction |
|
Gαi agonist assay
|
| Basal cAMP falls out of the lowest detection limit in the cAMP standard curve (i.e., cAMP level too low) | Insufficient number of cells | Increase the number of cells in the assay reaction |
| Forskolin-stimulated cAMP not achieving high enough level (i.e., cAMP level too low) | Not enough cells | Increase the number of cells in the assay reaction |
| cAMP rapidly degraded by endogenous | Include phosphodiesterase inhibitors such as IBMX |
| Forskolin concentration too low | Increase the concentration of forskolin as determined in forskolin concentration-response curves |
| Forskolin stimulated cAMP exceeded the linear part of the cAMP standard curve (i.e., cAMP level too high) | Too many cells | Decrease the number of cells in the assay reaction |
| Forskolin concentration too high | Reduce the concentration of forskolin as determined in forskolin concentration-response curves |
| Agonist-stimulated change in cAMP level undetectable | Cells not expressing sufficient quantities of receptor | Select for higher expressing clones as determined by qPCR or by FACS |
| Compounds not efficacious | Test other compounds known to be efficacious in modulating cAMP production |
| Insufficient compounds binding to the receptor | Allow for longer incubation of the assay reactions |
|
Gαs antagonist assay
|
| Antagonist compounds do not antagonize the agonist-stimulated response or not giving expected potency | Insufficient antagonist compounds binding to the receptor | Allow for longer pre-incubation of the antagonist compounds |
| Compounds not efficacious | Test other compounds known to be efficacious in modulating cAMP production |
| Agonist stimulation too strong | Reduce agonist concentration used for stimulation |
|
Gαi antagonist assay
|
| Antagonist compounds do not antagonize the agonist-stimulated response or not giving expected potency | Insufficient antagonist compounds binding to the receptor | Allow for longer pre-incubation of the antagonist compounds |
| Compounds not efficacious | Test other compounds known to be efficacious in modulating cAMP production |
| Agonist stimulation too strong | Reduce agonist concentration used for stimulation |
| Accumulated cAMP not degraded | Test presence or absence of phosphodiesterase inhibitor |
