| Assay Method Information | |
| | VNN1 Inhibitor Binding Assay |
| Description: | 5 μL of 3× (45 μM) of Pantothenate-AMC to achieve final concentrations of 15 μM (0.25% DMSO final) were added in a 384 well assay plate followed by the addition of 5 μL of 3× the desired concentration of the tested compounds (Compound 1; Compound 2; and RR6) in 0.75% DMSO to designated wells (0.25% of DMSO final). The range of the compound concentrations tested was 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.0046, 0.001, and 0.0005 μM. 5 μL of 0.75% DMSO were also added to positive and negative control wells.5 μL of 1.4 ng/μL Vanain-1 was added to the assay plate to achieve a final amount of 7 ng/well, and covered with plate sealer. Another 5 μL of assay buffer was added to negative controls and the assay plate was then centrifuged briefly (1000 rpm for 30 s) in a plate centrifuge. The assay plate was incubated in the dark for 20 minutes at room temperature and immediately read on an Envision 2105, PerkinElmer multimode plate reader.The fluorescence read was captured at excitation wavelength of 355 nm and emission wavelength of 460 nm. |
| Affinity data for this assay | |
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