| Assay Method Information | |
| | Quantification of cGAS Protein Inhibition |
| 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); and 0.001% 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; and 0.001% 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, [15N5]-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). The concentration of cGAMP was measured on a LC-MS/MS system consisting of a THERMO Dionex Ultimate LC system with a high pressure pump, an autosampler, a column heating compartment (Reinach, Switzerland) and a SCIEX Triple Quad 5500 (Framingham, MA, USA) mass spectrometer for detection. The sample plates were centrifuged for 10 minutes at 2000 rpm. Up to three plates were placed in the autosampler for injection. An aliquot of 10 μL of each sample was injected on an XBridge BEH Amide 3.5 μm, 2.1×50 mm column (P/N 186004859) with an XBridge BEH Amide 5 μm 2.1×5 mm VanGuard Cartridge (P/N 186007760) pre-column (both WATERS, MA, USA) held at 40° C. An isocratic flow of 1.0 mL/min solvent (60% ACN, 8 mM ammonium acetate, 5 mM ammonium hydroxide, 0.04% acetic acid) was applied and sprayed into the ion source of the mass spectrometers. The MS parameters were optimized based on the properties of the compounds to be detected and run in positive multi-reaction mode (MRM) based on the mass transitions. LC and MS parameters were also optimized to allow for a sample-to-sample measuring time of approximately 75 sec and a run time of 8 hours per 384-well plate. All data were analyzed with Excel; and the dose response curves were generated using the auto fitting function of XLfit. The IC50 was determined by plotting the cGAMP concentration ratio (cGAMP divided by the internal standard [15N5]-2′3′-cGAMP) versus the concentration of compound. |
| Affinity data for this assay | |
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