| Assay Method Information | |
| | Human LTC4S Enzymatic Assay |
| Description: | LTC4 synthase catalyzes the conversion of Leukotriene A4(LTA4) to Leukotriene C4 (LTC4) in the presence of reduced glutathione (GSH) as a co-substrate. For compound testing, compounds are delivered as 10 mM stock solutions in 90% DMSO in matrix tubes. From this, a 1:3 dilution dose response series is prepared with a starting concentration of 30 μM to 0.1 nM. For the enzymatic assay 97.5 nL of compound/DMSO solution is transferred to each well and 5 μL enzyme solution (assay buffer: 50 mM bis-tris propane pH 7.3, 250 mM NaCl, 10 mM MgCl2, 0.001% MGN3) is added to the wells. The final enzyme concentration in the assay is 0.75 nM. The enzyme compound mixture is incubated at room temperature for 15 minutes prior to the addition of 5 μL substrate solution. As the primary substrate LTA4 is a highly unstable intermediate of the arachidonic acid pathway, LTA4 is substituted for a more stable LTA4 methyl ester form (LTA4-Me) for the purposes of screening. A final substrate concentration of 400 μM GSH and 5 μM LTA4-Me is chosen. The LTA4-Me is obtained commercially in 2% triethylamine/hexane solvent. As this solvent is incompatible with the HTRF assay it has to be exchanged with DMSO according to following procedure: add 50 μL of 100% DMSO to 50 μL LTA4-Me (3 mM) in a 2 mL Eppendorf tube and mix gently by inverting the tube. The triethylamine/hexane is evaporated under a constant argon flux at room temperature. The DMSO-LTA4-Me (3 mM) is aliquoted and stored at −20° C. for not longer than 4 weeks, as it is not stable in DMSO due to its oxidizing properties. Upon addition of the substrate, the plate is immediately placed on a shaker for 5 min at room temperature. Immediately after the 5 min incubation 5 μL of H2O solution is added to all wells to stop the reaction. The plate contents are mixed before the addition of detection reagents. The conversion from LTA4-Me and GSH to LTC4-Me is quantified using a LTC4-Me standard curve ranging from 1.5 μM to 0.08 nM. For the detection of the product of the enzymatic reaction LTC4-Me, the Cisbio LTC4-HTRF kit is used as the assay is compatible with the detection of LTC4-Me. 5 μL of diluted LTC4-d2 conjugate (according to manufacturer's protocol) are added to all wells of the assay plate and the contents gently mixed and incubated for 5 minutes at room temperature. Then 5 μL of the diluted LTC4-Eu3+ cryptate (according to manufacturer's protocol) are added to all wells and the contents of the plate gently mixed and incubated 60 min at room temperature before reading the plate on the Spectramax Paradigm (Molecular Devices) using ratiometric analysis (665/616 nM) and the following setup: number of flashes/well of 30, integration time of 0.3 ms, excitation time of 0.05 ms, positioning delay of 0.03 ms, and a ratio multiplicator of 10000. |
| Affinity data for this assay | |
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