| Assay Method Information | |
| | MAGL Inhibitory Activity Assay |
| Description: | MAGL inhibitory activity by determining the enzymatic activity by following the hydrolysis of the natural substrate 2-arachidonoylglycerol (2-AG) resulting in arachidonic acid, which can be followed by mass spectrometry. This assay is hereinafter abbreviated “2-AG assay”. The 2-AG assay was carried out in 384 well polypropylene assay plates. Compound dilutions were made in 100% DMSO in a polypropylene plate in 3-fold dilution steps to give a final concentration range in the assay from 12.5 μM to 0.8 pM. Compound dilutions were added to MAGL protein in assay buffer (50 mM TRIS, 1 mM EDTA, 0.01% (v/v) Tween-20, 2.5% (v/v) DMSO). After shaking, the plate was incubated for 15 min at RT. To start the reaction, 2-arachidonoylglycerol in assay buffer was added. The final concentrations in the assay was 50 pM for MAGL protein and 8 pM 2-arachidonoylglyerol. After shaking and 30 min incubation at RT, the reaction was quenched by the addition of two assay volumes of acetonitrile containing 4 μM of d8-arachidonic acid. The amount of arachidonic acid formed was traced by an online SPE system (Agilent Rapidfire) coupled to a triple quadrupole mass spectrometer. |
| Affinity data for this assay | |
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