| Assay Method Information | |
| | Enzyme Assay |
| Description: | The assay was performed in a 100 μL final volume in 96-well UV plates (Costar, 3635) with a reaction buffer composed of 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 3 mM EDTA and 1 mM dithiothreitol, 4% v/v DMSO plus or minus inhibitory compound and 0.75 μg of purified IMPDH II enzyme per well (from 1 mg/mL stock concentration). The final volume of the enzyme stock solution per well was 0.75 μL which was insignificant to cause any change in the final assay buffer composition. The reaction was initiated by the addition of 300 μM of IMP and 500 μM of NAD+ and the assay was allowed to proceed at 37 °C for 50 min before stopping by the addition of 15 mM GMP. The NADH generated was measured by reading the absorbance at 340 nm. At this wavelength, a background of <0.1 optical density (OD) was observed with negligible crosstalk between wells. Molecular Devices Spectramax 384 plus (Molecular Devices, LLC, Sunnyvale, CA) was used for recording the absorbance, which allows for selection of up to six wavelengths at a time for absorbance detection in the UV-vis range (190-1000 nm). Mycophenolic acid (10 μM) used as a positive control and DMSO as a vehicle control. |
| Affinity data for this assay | |
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