| Assay Method Information | |
| | Scintillation Proximity (SPA) NLRP3 Assay |
| Description: | SPA experiments were conducted in a total volume of 20 μL assay buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.01% (v/v) Tween-20, 0.01% (w/v) bovine serum albumin (BSA) and 100 μM of adenosine diphosphate (ADP). A human NLRP3 protein (350 nM) (see, e.g., Sharif et al., Nature (2019) 570:338-343; Hochheiser et al., Nature (2022) 604:184-189; Adreeva et al., Cell (2021) 184:6299-6312; Wang et al., J. Exp. Med. (2021) 219:e20211147) was incubated with various concentration of compounds in 5% dimethyl sulfoxide (DMSO) final for 15 minutes at room temperature in a white half-area 96 well OptiPlate (PerkinElmer #6002290) before adding 125 nM final of 3H-MCC950. After the addition of radiolabeled MCC950, the plate was incubated for 60 minutes at room temperature before adding 0.25 mg/ml of copper-coated polyvinyltoluene SPA beads (PerkinElmer #RPNQ0095). The plate was then transferred to a microplate counter (PerkinElmer 2450 Microbeta) to collect binding data. Binding data was analyzed 6 hrs post-PVT bead addition to allow sufficient time to reach equilibrium. Specific binding was calculated from averaged duplicates by subtracting signal from wells without NLRP3 protein. |
| Affinity data for this assay | |
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