| Assay Method Information | |
| | ThermoFluor Assay |
| Description: | The ThermoFluor (TF) assay is a 384-well microplate-based binding assay that measures thermal stability of proteins1,2. The experiments were carried out using ThermoFluor instruments available from Johnson & Johnson Pharmaceutical Research & Development, LLC. TF dye used in all experiments was 1,8-ANS (Invitrogen: A-47). Final TF assay conditions used for MGL studies were 0.07 mg/ml of mutant MGL, 100 uM ANS, 200 mM NaCl, 0.001% Tween-20 in 50 mM PIPES (pH=7.0).Screening compound plates contained 100% DMSO compound solutions at a single concentration. For follow-up concentration-response studies, compounds were arranged in a pre-dispensed plate (Greiner Bio-one: 781280), wherein compounds were serially diluted in 100% DMSO across 11 columns within a series. Columns 12 and 24 were used as DMSO reference and contained no compound. For both single and multiple compound concentration-repsonse experiments, the compound aliquots (46 mL) were robotically predispensed directly into 384-well. |
| Affinity data for this assay | |
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