UA-Glo® Nano-Steady Luciferase Assay System

The luciferase-based reporter gene system is widely used in studies on intracellular signaling pathways, transcription factor regulation, receptor functions, high-throughput drug screening, etc. The expression of intracellular luciferase can be quantitatively detected. Its working principle is that luciferase catalyzes the substrate, causing a transformation that produces spontaneous cold light, and the intensity of the luminescence is positively correlated with the amount of luciferase.

The Nano-Steady Luciferase Detection Kit is a homogeneous ready-for-use reagent that integrates cell lysis and luciferase detection. Through the "add-mix-detect" process, it eliminates the experimental step of detecting after lysis first. Compared with the traditional Steady Luciferase assay system, the Nano luciferase molecule is smaller, less susceptible to non-specific effects of screening compounds, and at the same time, the detection reaction reading is significantly improved, so the detection sensitivity is also significantly enhanced. The cells to be detected are transfected with plasmids containing the Nano luciferase reporter gene.

After mixing the buffer with the substrate, it is filled into 10 ml or 100 ml brown bottles and 2 ml screw-cap tubes. The specifications are as follows:

1. Seed the cells to be tested at an appropriate density in a 96-well or 384-well cell culture plate, such as cells expressing a micro-luciferase reporter gene or cells expressing two proteins with micro-luciferase large and small fragments respectively. It is recommended to use a white plate.
2. Perform relevant treatments on the cells according to the project requirements and continue culturing for an appropriate period of time.
3. Take out the detection lysis buffer and equilibrate it to room temperature. Centrifuge the substrate tube quickly for 10 seconds to collect the contents at the bottom of the tube, and place it on an ice box. Take out the cell culture plate to be tested and equilibrate it to room temperature.
4. Prepare 1x detection working solution: Determine the amount of reagent to be prepared according to the experimental needs. When preparing, add the substrate to the lysis buffer at a 200X ratio and mix thoroughly.
5. Add an equal volume of 1x detection working solution to the cells in the 96-well or 384-well plate. For example, add 100 μL of 1x detection working solution to 100 μL of culture medium, shake the plate to mix, and place it in the dark to continue lysis for at least 3 minutes.
6. Read the fluorescence signal on a fluorescence microplate reader. Save the data and perform data processing and analysis.

1.  Dosage: It is not recommended to arbitrarily change the dosage of reaction reagents without strict verification.

2.  If the expression level of micro-luciferase is low, before adding 1x detection reagent, the cell culture medium can be replaced with an equal volume of serum-free medium to obtain a better signal-to-noise ratio.

3.  It is not recommended to mix reagents from different batches.

4.  Aliquot storage: Aliquot and store the reaction reagents according to the instructions to ensure the stability of the reagents.