UA-Glo® NAD(P)H Detection System

NADH and NADPH (collectively referred to as NAD(P)H) are reactants or products in many enzymatic reactions, and the quantitative measurement of NAD(P)H can directly or indirectly determine the activity of numerous enzymes. The UA-Glo® NAD(P)H Detection Kit utilizes a coupled reaction of reductase and luciferase, where reductase equivalently consumes NADH or NADPH as substrates. Through NAD(P)H, the reductase substrate is converted into luciferin, which is then acted upon by luciferase to generate luminescence. The luminescence intensity is proportional to the amount of NAD(P)H consumed or produced in the enzymatic reaction in vitro, and this coupled reaction does not detect or is affected by the oxidized form of NAD(P).

The UA-Glo® NAD(P)H Detection Reagent is a homogeneous reagent, requiring only a single addition for detection, thereby reducing experimental errors caused by multiple operations. This reagent features high signal-to-noise ratio, excellent reproducibility, and stability. Its stable glow signal (half-life >2 hours) makes it particularly suitable for high-throughput screening of enzyme inhibitors.

UA-Glo® NAD(P)H Detection Kit components and specifications are as follows. Based on an enzymatic reaction volume of 50μL for a 96-well plate and 25μL for a 384-well plate, 10mL allows for 200 reactions (96-well plate) or 400 reactions (384-well plate), while 50mL allows for 1,000 reactions (96-well plate) or 2,000 reactions (384-well plate).

Specification	Luciferin Detection Reagent	Reductase	Reductase Substrate
10mL	10 mL	200 μL	50 μL

Reagent Preparation

UA-Glo® NAD(P)H detection reagent should be freshly prepared as needed. It is not recommended to store pre-prepared detection reagent. During detection, an equal volume of detection reagent as the enzymatic reaction volume should be added. Calculate the required volume of detection reagent accordingly.

1)Take out the three components from the UA-Glo® NAD(P)H detection kit.

2)After thawing the luciferin detection solution at room temperature, it can be kept at room temperature (22-25°C) until use(not exceeding24hr), or stored at4°Cfor up to one week. For long-term storage, aliquot and store at-20°Cor lower temperatures, avoiding repeated freeze-thaw cycles.

3)The reductase concentration is50x. After thawing, add the required volume (see step5) to the luciferin detection solution. The remaining reductase should be aliquoted and stored at-20°Cor lower temperatures, avoiding repeated freeze-thaw cycles.

4)The reductase substrate concentration is200x. After thawing, add the required volume (see step5) to the luciferin detection solution.The remaining reductase substrate should be aliquoted and stored at-80°C, avoiding repeated freeze-thaw cycles.

5)NAD(P)Hdetection reagent preparation: The reductase concentration is50x, and the reductase substrate concentration is200x. Mix the two components into the luciferin detection solution at this ratio to prepare theNAD(P)Hdetection reagent. For example, add20μLof reductase and5μLof reductase substrate to1mLof luciferin detection solution. Invert the reagent to mix. It is recommended to use the preparedNAD(P)Hdetection reagent immediately.

NAD(P)H Detection

1)Perform enzymatic reactions that consume or generateNAD(P)H)according to experimental requirements:

a)It is recommended to use white opaque assay plates for enzymatic reactions. The reaction volume for a 96-well plate is50μL, for a 384-well plate is25μL, or other appropriate enzymatic reaction volumes. During detection, add an equal volume ofUA-Glo® NAD(P)Hdetection reagent as the enzymatic reaction volume.

b)Avoid reducing agents such asDTTin enzymatic reactions. Reducing agents may react with the reductase substrate and increase detection background.

2)BeforeNAD(P)H detection, equilibrate the assay plate to room temperature. The luciferase reaction is sensitive to temperature changes. Both the detection reagent and the assay plate need to be equilibrated to room temperature, and the temperature should remain constant during testing (±1℃).

3)Add an equal volume ofUA-Glo® NAD(P)Hdetection reagent as the enzymatic reaction volume to each well of the assay plate.

4)Shake the plate at medium speed for30s, then incubate in the dark for40-60minto stabilize the luminescent signal.

5)Read the luminescent signal on a luminometer or multifunctional plate reader. The plate can be read within2-3hr. The luminescent signal typically peaks at60min, with a signal half-life of>2hr.NAD(P)Hdetection reagent stops most enzymatic reactions upon addition. However, some enzymes may continue to react after the addition of the detection reagent. If a stable signal is required, add a specific inhibitor for such enzymes to theNAD(P)Hdetection reagent before adding it to the enzymatic reaction for detection.

1) Different batches are not recommended for mixed use 2) Without strict validation, it is not recommended to change the dosage of detection reagents 3) For research use only