| Assay Method Information | |
| | cAMP Assay |
| Description: | Cells were seeded at a density of 10,000 cells per well in poly-L-lysine treated 96-well culture plates (BD Biosciences). The following day wells were incubated with 40 μl DMEM/F12 containing 0.5% (w/v) BSA and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 30 minutes prior to 15 min stimulation with 50 μM forskolin (Tocris Cookson) and varying concentrations of indicated compounds at 37° C., 5% CO2. Assays were stopped by removal of media and addition of 100% ice cold ethanol. Plates were then frozen for a minimum of two hours before complete evaporation of ethanol. The well contents were then reconstituted in 50 μl cAMP assay buffer (20 mM HEPES pH 7.5 and 5 mM EDTA). Half of the reconstituted sample was transferred to round bottom 96-well plates (Greiner Bio-One GmbH) with 50 μl 0.01% w/v PKA (cAMP dependent protein kinase (Sigma-Aldrich) in 1 mM Na citrate pH 6.5 with 2 mM dithiothreitol) and 25 μl [3H]-cAMP (at 22 nM in cAMP assay buffer) (GE Healthcare, Life Sciences). This was allowed to equilibrate for 3-18 hours. Following this a charcoal slurry (5% (w/v) activated charcoal and 0.2% (w/v) BSA in cAMP assay buffer) was added to the samples and the plates centrifuged at 3000×g, 4° C. for 5 min. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |