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| | RF-MSS cyclase assay |
| Description: | The RapidFire 365 High-throughput MS System (Agilent Technologies; RF-MSS) can process samples every 15 seconds allowing analysis of a 384 well plate in under two hours; thus, RF-MSS provides a platform for high throughput MS screening which can simultaneously measure both cAMP produced and ATP consumed in an individual sAC assay. Using the RF-MSS we observed no appreciable sample carryover between assay samples, and we were able to detect both a sAC-dependent increase in cAMP signal (FIG. 2A) and decrease in ATP signal (FIG. 2B) over time. When measured in the presence of increasing concentrations of substrate ATP in the presence of Mg2+ as the sole divalent cation, HCO3 − stimulated sAC activity by increasing the Vmax with little effect on the apparent Km for substrate ATP-Mg2+ (FIG. 2C). Addition of Ca2+ stimulated sAC activity by decreasing its apparent Km for ATP, and addition of both Ca2+ and HCO3 − synergistically activated sAC. These data confirm that using RF-MSS as a tool for measuring in vitro adenylyl cyclase activity is comparable to the known, radioactivity based, two-column method for measuring adenylyl cyclase activity. |
| Affinity data for this assay | |
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