| Assay Method Information | |
| | In Vitro Inhibition Assay |
| Description: | In vitro inhibition assay of cGAS activity: Add a 5 μL mixture containing 10 mM Tris-Cl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% Triton X-100, 60 nM 75-bp dsDNA, 501.1M ATP, 50 μM GTP, 10 nM of recombinant human cGAS, and serial dilutions of a test compound in DMSO to a 384-well plate and incubate at room temperature for 3 hours. Quench the reaction by the addition of 100 μL of stop solution containing 0.1% formic acid and 0.1 μM internal standard (3′3′-difluoro cGAMP). Measure the amount of cGAMP produced by RapidFire 365 mass spectrometry and evaluate inhibitory effect of a compound by plotting percent specific inhibition of cGAMP production against log concentration of the test compound. |
| Affinity data for this assay | |
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