| Assay Method Information | |
| | Biochemical Assay |
| Description: | A reagent buffer was prepared in filtered and autoclaved water according to the following:50 mM Tris-buffer pH 7.5 (1 M Tris-buffer pH 7.5, Invitrogen, Cat. No. 15567-027);50 mM NaCl (5 M NaCl, Sodium Chloride Solution, Sigma, 59222C-);5 mM MgCl2 (1 M MgCl2, Sigma, M1028);0.1 mM ZnCl2 (Zinc Chloride [7646-85-7], powder, Cell Culture Tested, Sigma, Z-0152); and0.001% Tween 20 (TWEEN 20, Sigma Aldrich, P1379-).A buffer for the cGAS enzyme was prepared in filtered and autoclaved water according to the following:50 mM Tris-buffer pH 7.5;5 mM MgCl2; and0.001% Tween 20.Compounds were dispensed to a 386 well plate. The human truncated cGAS enzyme (4.2 mg/mL 147-522 human cGAS, MW 43,909 g/mol) was stored in 50 mM Tris, 500 mM NaCl, 5% (v/v) glycerol at pH 8 and diluted in the cGAS buffer enzyme shortly before use. The enzyme solution was transferred into the reagent buffer to give a final concentration of 30 nM. The reaction was started by mixing the enzyme with ISD (a 45 bp double stranded DNA, MW 27,670 g/mol, 5 mM), GTP and ATP to a final concentration of 5 μM, 0.5 mM and 0.5 mM respectively in a final volume of 10 μl. The reaction plates were then centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 1 h. After 1 h of incubation, [5Ns]-2′3′-cGAMP to a final concentration of 200 nM and 30 μL of 100% acetonitrile/0.175% of TFA were added to the reaction mixture. The plates were centrifuged at 1000 rpm for 1 minute before being sealed for 3 seconds at 170° C. using a ThermoScientific sealer (ALPS™ 50V) and an aluminum sealing cover (Pierce Seal, 4titude, Product Code: 4TI-0531). |
| Affinity data for this assay | |
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