| Assay Method Information | |
| | Enzymatic Assay |
| Description: | Method 1:Vanin-1 Enzymatic Assay:The test compounds are dissolved in 100% DMSO at a concentration of 10 mM and in a first step diluted in DMSO to a concentration of 5 mM, followed by serial dilution steps in 100% DMSO. Dilution factor and number of dilution steps may vary according to needs. Typically 8 different concentrations by 1:5 dilutions are prepared, a further intermediate dilutions of the substances is carried out with assay buffer resulting in 1% final DMSO concentration in the assay. 0.15 nM of FLAG-tagged Vanin-1 (AA 22-493, T26I, produced internally) and test compounds are incubated at room temperature for 15 minutes in assay buffer (1 mM DTT, 0.0025% Brij-35, 50 mM HEPES, pH7.5). 3 μM D-Pantethine (Sigma, Cat#P2125-5G) in assay buffer is added and incubated for additional 30 minutes at room temperature. Reaction is stopped by adding equal volume of stop solution as the reaction mixture to reach 5 nM Nevirapine (as an internal standard) and 0.5% TFA. Assay plates are centrifuged for 2 minutes and the formation of pantothenic acid is detected by RapidFire Mass Spectrometry (mobile phase A: 0.1% formic acid in water, mobile phase B: 100% methanol) using graphitic carbon cartridge (Agilent Cat #G9206A). |
| Affinity data for this assay | |
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