| Assay Method Information | |
| | MAGL Inhibitory Activity Assay |
| Description: | The asssay is hereinafter abbreviated "2-AG assay". The 2-AG assay was carried out in 384 well assay plated (PP, Greiner Cat #784201) in a total volume of 20 uL. Compound diutions were made in 100% DMSO (VWR Chemicals 23500.297) in a polypropylene plate in 3-fold dilution steps to give a final concentration range in the assay from 12.5 uM to 0.8 pM. 0.25uL compound dilutions (100% DMSO) were added to 9 uL MAGL in assay buffer (50 mM TRIS (GIVCO, 15567-027), 1 mM EDA (Fluka, 03690-100 nK), 0.01% (v/v) Tween. After shaking. the plate was incubated for 15 min at RT. To start the reaction, 10 uL 2-arachidonoylglycerol in assay buffer was added. The final concentrations in the assay was 50 pM MAGL and 8 uM 2-arachidonoylglycerol. After shaking and 30 min incubaction at RT, the reaction was quenched by the addition of 40 L of ACN containing 4 uM of d8-arachidonic acid. The amount of arachidonic acid was traced by an online SPE system (agilent Rapidfire) coupled to a triple quadrupole mass spectrometer (Agilent 6460). A C18 SPE cartridge (G9205A) was used in an ACN/water liquid setup. The mass spectrometer was operated in negative electrospray mode following the mass transitions 303.1-259.1 for arachidonic acid and 311.1-267.0 for d8-arachidonic acid. |
| Affinity data for this assay | |
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