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| | Enzymatic Activity Assay |
| Description: | This assay measures the substrate consumption by quantifying the remaining NAD+ through its chemical conversion to a stable fluorescent condensation product upon treatment by acetone and alkaline followed by heating in acidic conditions. Reaction was carried out on U-shaped black polypropylene 96-well plates (Greiner BioOne, Frickenhausen, Germany). The volume in the control (maximal signal) and reaction (minimal signal) wells was 50 µL, and they contained buffer and NAD+ (Sigma-Aldrich) (500 nM in most experiments) or buffer, NAD+, and tankyrase 1, respectively. The plate was kept at 25 °C, 300 rpm using the Biosan PST 100HL (Riga, Latvia) plate shaker during the enzymatic reaction. The plate was covered with an Amplate cover (Simport, Waltham, MA) throughout the enzymatic and chemical reaction. Original assay protocol was modified so that 20 µL of 20% acetophenone in ethanol was added in the fume hood before the addition of 20 µL 2 M KOH. After incubation, the plate was moved to 4 ° |
| Affinity data for this assay | |
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