| Assay Method Information | |
| | LP-PLA2 SAR Assay (0.2 nM) |
| Description: | I. Recombinant Human Lp-PLA2 (hLp-PLA2) Enzymatic Assay Using PED6 (Invitrogen) as Substrate[0439]Recombinant hLp-PLA2 (0.2 nM final concentration) was preincubated at room temperature with compounds for 20-30 mins. Reactions were then initiated upon the addition of substrate solution containing 2 uM PED6. The resulting fluorescence intensity change was monitored kinetically for 20 mins using a Tecan Safire 2 at FLINT 480/540 or Perkin-Elmer Envision at FLINT 480/530.The pIC50 value (negative log of the IC50 value when converted to molar) results shown in Table 2 for compound 1218-39, 1218-20, 1218-20S, 1218-20R, 1218-40, 1218-4A, 1218-41B, 1109-15, 1109-14, 1109-51, 1109-52, 1109-52S, 1109-55 and 1109-55S were at least 8.72 for Enzymatic assay for 0.2 nM concentration, and at least 8.27 for Enzymatic assay for 2 nM concentration.II. Human Plasma Lp-PLA2 Assay Using 2-Thio-PAF as Substrate 8 μL human plasma was pre-incubated with compound for 30 mins at room temperature. The reaction was initiated by adding 2 μl substrate working solution, which containing 2.5 mM 2-thio-PAF (Cayman Chemical), 32 uM CPM (Invitrogen) and 3.2 mM NEM (Thermo). After 2 mins, 5 μl quench solution (5% TFA) was added to stop the reaction. Plates were then settled for 40 mins and centrifuged for 1 min at 2000 rpm. Assay plates were read for FLINT signal on Perkin-Elmer Envision (FLINT 380/485). |
| Affinity data for this assay | |
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