NAMPT Activity Assay Kit (Colorimetric)

Product details

NAMPT Activity Assay Kit (Colorimetric) (ab221819) provides a sensitive and robust method to evaluate activators and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) activity using recombinant NAMPT protein. The assay can also be used to measure activity from endogenous NAMPT immunoprecipitated from cell extracts. The assay is based on a multi-step reaction that converts WST-1 to WST-1 formazan, which can be easily detected at OD 450 nm. As the reaction is not stopped, it is necessary to monitor the absorbance increase of WST-1 formazan at OD450 nm at regular intervals after the reaction is initiated to determine velocity of reaction.

Detection of NAMPT activity can be measured with a One-Step or a Two-Step method. For the One-Step method, the four enzymes involved in the reaction are mixed together. The detection sensitivity of this method is lower than that of the Two-Step method since three coupled reactions occur simultaneously. We recommend performing the One-step assay only when using purified protein. The Two-Step method is performed by adding the first two enzymes to produce NAD⁺, followed by the addition of the rest of the components to form WST-1 formazan by NAD/NADH enzyme cycling reaction. We recommend performing the Two-Step assay if using immunoprecitated cell lysates, as the assay procedure allows to check for contamination and interference for NAD⁺ present in the sample. Since the assay is based on a NAD⁺ detection system, it is not possible to directly detect NAMPT activity in crude cell extracts, which contain relatively high NAD⁺.

For the One-Step method, we describe a procedure to test NAMPT activity using purified enzyme and a separate procedure for screening NAMPT activity inhibitors. For the Tw-Step Method, we describe two different procedures to test NAMPT activity using purified enzyme or to test immunoprecipitated cell lysates. There is also a separate procedure for screening NAMPT activity inhibitors.

Other Notes
Nicotinamide phosphoribosyltransferase (NAMPT, visfatin, pre-B-cell colony-enhancing factor, EC 2.4.2.12), is the rate-limiting enzyme that converts nicotinamide to nicotinamide mononucleotide (NMN) in the NAD⁺ salvage pathway of NAD⁺ biosynthesis in mammals. The expression of NAMPT is upregulated during activation of immune cells such as monocytes, macrophages, dendritic cells, T and B cells, as well as in amniotic epithelial cells upon stimulation with several inflammatory cytokines. The NAMPT-specific inhibitor FK866 has been found to deplete intracellular NAD content and result in apoptotic cell death in many cancer cell lines without any DNA damage.