| Assay Method Information | |
| | Fluorescence Based Assay |
| Description: | The assay buffer "AB" contained 50 mM ADA (N-(2-acetamido)iminodiacetic acid monosodium salt) pH 6.5, 1 mM dithiothreitol, 0.006% Triton-X100 and 50 mM NaCl. The following components are added in a white polystyrene Costar plate (Ref 3912) up to a final volume of 55.5 uL: 1.5 uL DMSO or inhibitor dissolved in DMSO and 54 uL of a FabI/NADPH/NADP+ mixture in AB. After 60 min of pre-incubation at room temperature, the reaction is started by addition of 5 uL of trans-2-octenoyl N-acetylcysteamine thioester (t-o-NAC) to a final volume of 60.5 uL. This reaction mixture is then composed of 2 nM FabI, 40 uM NADPH (Sigma, N7505), 10 uM NADP+(Sigma, N5755), 100 uM t-O-NAC and compound at defined concentration. Fluorescence intensity of NADPH (lamda=ex=360 nm, lamda=em=520 nm) is measured immediately after t-O-NAC addition (T0), and approximately 50 min later (T50) by a Fluostar Optima (BMG) so as to achieve ~30% of NADPH conversion. |
| Affinity data for this assay | |
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