| Assay Method Information | |
| | GCL Enzyme Profiling Assay |
| Description: | The GCL enzyme assay was run in 50 mM HEPES, pH 7.5, containing 150 mM NaCl, 30 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.1 mg/mL BSA, and 0.02% or in the present case 0.2% F-127 in a total assay volume of 10 μL or 10 mL. Compound in pure DMSO was stamped in assay plates (Greiner black 384-well) or appropriate containers using a D300e digital dispenser (TECAN) and the final DMSO concentration was normalized to 1%. A 5 mL, or in the present case, 5 μL mixture of human recombinant GCL (final concentration: 5 nM) and ATP (final concentration: 0.6 mM) was added and the solution was incubated at room temperature (22° C.) for 1 h. The GCL enzyme reaction was initiated by addition of a 5 μL mixture of mono g-glutamate or in the present example, monosodium glutamate (final concentration: 1 mM) and a-aminobutyrate or α-aminobutyrate (final concentration: 1.2 mM). The reaction was stopped after 1 h or in the present case, 2 h by addition of 5 mL, or in the present case, 5 μL ADP GLO Reagent (Promega). The resulting solution was incubated for 2 h at room temperature. This was then followed by addition of 5 mL, or in the present case, 5 μL ADP GLO Detection. After incubation at room temperature for 30 min, the plates were read on a plate reader (PheraStar) using a luminescence protocol to quantify the GCL enzyme activity from each reaction solution. |
| Affinity data for this assay | |
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