Amplite® Luminometric Nitroreductase Assay Kit

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H-phrase	H303, H313, H333
Hazard symbol	XN
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R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Nitroreductases (NTR) are a family of evolutionarily related proteins involved in the reduction of nitrogen-containing compounds. They are absent in mammalian cells but widespread in bacteria. Nitroreductases play a crucial role in the reduction of nitroaromatic compounds via NAD(P)H-dependent reactions. NTR are also targets for developing novel antibiotics, degradation of pollutants, and cancer therapy. Although the quantification of NTR becomes extremely important for a number of biological applications, most of the current NTR assays are based on ELISA format with low sensitivity and low throughput. Amplite®™ Luminometric Nitroreductase Assay Kit provides a highly sensitive and convenient method to quantify NTR activity. The kit uses a luciferin derivative that can be selectively reduced by NTR luciferin. The amount of generated luciferin is conveniently detected with the well-known luciferase detection system. The luminescent intensity generated is proportional to the NTR activity. This luminometric nitroreductase assay kit has been formulated to have minimal hands-on time and stable luminescence signal. It can detect as low as 20 ng/mL of NTR.

Platform

Luminescence microplate reader

Recommended plate

Solid white

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare NTR Substrate stock solution
Add NTR standards or NTR test samples (50 µL/well)
Add NTR Reaction working solution (50 µL/well)
Incubate for 60 minutes at 37 °C
Prepare NTR Detection working solution
Add NTR Detection working solution (50 µL/well)
Monitor luminescence intensity increase immediately

Important

Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NTR substrate stock solution (100X)

Add 50 µL of DMSO (Component H) into the vial of NTR Substrate (Component A) to make 100X NTR substrate stock solution.
Note Keep away from light.

2. NTR Reaction Enzyme stock solution (100X)

Add 50 µL of NTR Reaction Buffer (Component B) into the vial of NTR Reaction Enzyme (Component C) to make 100X NTR Reaction Enzyme stock solution.
Note Keep away from light.

3. NTR Detection Mix 1 stock solution (100X)

Add 50 µL of NTR Detection Buffer (Component D) into the vial of NTR Detection Mix 1 (Component E) to make 100X NTR Detection Mix 1 stock solution.
Note Keep away from light.

4. Nitroreductase Standard solution (250 µg/mL)

Add 20 µL of ddH₂O into the vial of Nitroreductase Standard (Component G) to make 250 µg/mL Nitroreductase Standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/12470

Nitroreductase Standard solution

Add 10 µL of 250 µg/mL Nitroreductase Standard solution to 240 µL of NTR Reaction Buffer (Component B) to generate 10 µg/mL nitroreductase standard solution (NS7). Then take 10 µg/mL nitroreductase standard solution (NS7) and perform 1:3 serial dilutions in NTR Reaction Buffer (Component B) to get serially diluted Nitroreductase standards (NS2 - NS7). Note: Diluted NTR standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

1. NTR Reaction working solution

Add 50 µL of 100X NTR substrate stock and 50 µL of 100X NTR reaction enzyme stock solutions into 5 mL of NTR Reaction Buffer (Component B) to make a total volume of 5.1 mL NTR Reaction working solution.
Note Keep away from light.

2. NTR Detection working solution

Add 50 µL of 100X Detection Mix 1 and 10 µL of Detection Mix 2 (Component F) into 5 mL of NTR Detection Buffer (Component D) to make a total volume of 5.05 mL NTR Detection working solution.
Note Keep away from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1.Layout of NTR standards and test samples in a solid white 96-well microplate. NS=NTR standards (NS7-NS1, 0.001 to 10 µg/mL); BL=Blank Control; TS=Test Samples

BL	BL	TS	TS
NS1	NS1	...	...
NS2	NS2	...	...
NS3	NS3
NS4	NS4
NS5	NS5
NS6	NS6
NS7	NS7

Table 2.Reagent composition for each well

Well	Volume	Reagent
NS1-NS7	50 µL	Serial Dilutions (10 to 0.001 µg/mL)
BL	50 µL	NTR Reaction Buffer (Component B)
TS	50 µL	Test Sample

Prepare NTR standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 12.5 µL of reagent per well instead of 50 µL.
Note Treat cells or tissue samples as desired.
Add 50 µL of NTR Reaction working solution to each well of NTR standard, blank control, and test samples. For a 384-well plate, add 12.5 µL of NTR Reaction working solution into each well instead.
Incubate the reaction for 60 minutes at 37 °C, protected from light.
Add 50 µL of NTR Detection working solution to each well to make the total assay volume 150 µL/well. For a 384-well plate, add 12.5 µL of Detection working solution into each well instead, for a total volume of 37.5 µL/well.
Monitor the luminescence increase immediately with a luminescence microplate reader.

Images

Figure 1. Nitroreductase dose response was measured in a white solid 96-well plate with Amplite® Luminometric Nitroreductase Assay Kit using with a NOVO star plate reader (BMG Labtech). The kit can detect as low as 20 ng/mL of NTR (The data was obtained about 5 min after adding the NTR detection working solution).