| Assay Method Information | |
| | NAMPT Enzyme Activating Effect (In Vitro Cell-Free Enzyme Assay |
| Description: | The NAMPT enzyme assay was conducted in accordance with the method of Formentini et al. (Formentini L. et al., Biochemical Pharmacology 77 (2009) 1612-20) by chemically converting nicotinamide mononucleotide (NMN) produced by the NAMPT enzyme to a fluorescent substance and using the fluorescence intensity of this fluorescent substance as an index for the amount of NMN produced. Hereinafter, the procedures will be briefly described. The NAMPT enzyme reaction was carried out using polypropylene 384 well V shaped black plate (Greiner Bio One International GmbH). The NAMPT activity was measured in assay buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.1% Prionex, 0.005% Tween 20, 0.12 mM adenosine triphosphate (ATP), 5 μM nicotinamide (NAM), 6.25 μM phospho-ribosyl pyro-phosphate (PRPP), 0.04 U/mL pyrophosphatase and 2 ng/mL human NAMPT enzyme in the presence or absence of test compounds. After the 1-h incubation at 25° C., the enzyme reaction was terminated by the addition of 5 μL of 2 M KOH and 5 μL of a 20% acetophenone solution. Then, 22 μL of 88% formic acid was added and the mixture was further incubated for 30 min in the dark. The NAMPT enzyme activity was calculated as the difference between fluorescence intensity (Ex 380 nm/Em 450 nm) from the reaction of a test compound treatment group and fluorescence intensity from control reaction free from the NAMPT enzyme.[NAMPT enzyme activity]=[Fluorescence intensity of the test substance treatment group]−[Mean fluorescence intensity of the control reaction free from the NAMPT enzyme] |
| Affinity data for this assay | |
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