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| | TbrPDEB1 Enzyme Assay |
| Description: | In short, recombinant TbrPDEB1 and TbrPDEB2 were assayed in 10 mM Tris pH 7.4, bovine serum albumin (0.2 mg/mL), 10 mM MgCl2, 25µM cAMP (Enzo Lifesciences) and an excess of 5 endonucleotidase (>1000U) at 37 °C. Excess amount of 5 endonucleotidase was determined by titration (Enzo Lifesciences). Reactions were terminated by the addition of BioMol green (Enzo Lifesciences) which was also used to detect changes in the level of phosphate. This was measured by absorbance at 620 nm using a Tecan Sunrise plate reader. All screens were carried at a final concentration of 2% (v/v) DMSO and all IC50 experiments were carried out at 10% (v/v) DMSO. Inhibitors were preincubated with the assay mixture for 5 min prior to the addition of substrate. Inhibition was determined by the change in the initial velocity relative to a vehicle only control for both compound screening and for the determination of IC50 values. Initial velocity was determined by linear regression and IC50 values were calculated |
| Affinity data for this assay | |
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