NMNAT1 Activity Assay Kit (Colorimetric)

Product details

NMNAT1 Activity Assay Kit (Colorimetric) (ab221820) provides a sensitive and robust method to evaluate activators and inhibitors of nicotinamide mononucleotide adenylyltransferase (NMNAT) activity using recombinant NMNAT protein. The assay can also be used to measure activity from endogenous NMNAT 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 NMNAT 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 immunoprecipitated 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 NMNAT activity in crude cell extracts, which contain relatively high NAD⁺.

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

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1, EC 2.7.7.1) is a central enzyme in NAD+ biosynthesis, transferring the adenylyl moiety of ATP to β-Nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), resulting in the formation of NAD⁺ or NaAD and the release of pyrophosphate. This enzyme could be a potential target for therapeutical applications, because its activity is rather low in tumor cells. It has been shown that NMNAT1 can inhibit recombinant human poly(ADP-ribose) polymerase-1 (PARP-1) by about 35% and completely prevent the formation of branched ADP-ribose polymers.