Nano-Steady Luciferase Assay Kit Signal Stability Test: HEK293 cells at a density of 6 × 10^5/mL were seeded into a 6-well cell culture plate; 24 hours later, they were transfected with a plasmid containing the Nano luciferase reporter gene. Forty-eight hours post-transfection, the cells were transferred to a 96-well plate at 15,000 cells per well. After the cells had adhered, they were allowed to equilibrate at room temperature for 10 minutes, then the Nano-Steady luciferase assay reagent, pre-equilibrated to room temperature, was added. Luminescence values were continuously recorded at different time points.
UA-Glo® Nano-Steady Luciferase Assay System
UA-Glo® Nano-Steady Luciferase Assay System
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Product Specification
| Synonyms | Nano-Steady 荧光素酶检测试剂盒 |
| Stability & Storage | Dry ice transportation. Store at -20°C or below, protected from light. The expiration date can be found on the reagent bottle label. After initial use, it is recommended to aliquot the reagent as needed and store it at -20°C or below, protected from light. For long-term storage (>2 months), the substrate is recommended to be stored at -80°C. |
Background
The luciferase reporter gene system (Luciferase-based reporter gene system) is widely used in intracellular signaling pathways, transcription factor regulation, receptor function studies, and high-throughput drug screening. The expression of intracellular luciferase can be quantitatively detected. Its working principle involves the catalysis of substrates by luciferase, leading to transformation and the generation of spontaneous luminescence, with the intensity of luminescence being directly proportional to the amount of luciferase.
The Nano-Steady Luciferase Assay Kit is a homogeneous ready-for-use reagent that integrates cell lysis and luciferase detection into a single step, enabling a "add-mix-detect" workflow that eliminates the need for separate lysis and detection steps. Compared to traditional Steady Luciferase Assay systems, the Nano luciferase molecule is smaller and less susceptible to nonspecific interference from screening compounds, while also significantly improving detection signal readings. Consequently, the detection sensitivity is markedly enhanced. The cells being tested are those transfected with plasmids containing the Nano luciferase reporter gene.
Components
UA-Glo® The components and specifications of the stable micro-luciferase assay kit are as follows. Detectable96-well and384-well plate reaction counts are calculated based on samples of100μLand20μL, with100μLand20μLtest reagent calculation.
Specifications |
Lysis buffer |
Micro-luciferase substrate |
Detection96-number of wells in a plate |
detection384-Number of wells in a plate |
10 mL |
10 mL |
50μL |
100 |
500 |
100 mL |
100 mL |
0.5 mL |
1,000 |
5,000 |
10*10 mL |
10 x 10 mL |
10 x 50 μL |
1,000 |
5,000 |
10*100 mL |
10 x 100 mL |
10 x 0.5 mL |
10,000 |
50,000 |
Protocol
The luciferase reaction exhibits extremely high sensitivity, and parameters such as pH, temperature, and experimental system settings significantly influence the reaction. In most experimental conditions, direct comparison of absolute detection values between samples is not recommended. It is advised to include the same reference compound and solvent vehicle control on each experimental plate, and normalize the detection values of experimental samples using the reference compound and solvent vehicle control before data processing and comparison.
The following experiment uses the detection of intracellular nano-luciferase expression as an example.UA-Glo® stable nano-luciferase detection kit can also be used to detect the expression of secreted nano-luciferase outside cells. Since nano-luciferase is highly stable in culture medium, continuous detection of secreted nano-luciferase is possible. For example, each time 5-20 μL of culture medium containing secreted nano-luciferase can be transferred to a white assay plate, diluted to 100 μL with sterile ultrapure water, and detected following steps 4-8 below.
1.Seed cells expressing the nano-luciferase reporter gene at an appropriate density in a 96-well or 384-well white transparent cell culture plate.
2.Perform relevant treatments on the cells according to experimental requirements and continue culturing for an appropriate time until detection.
3.Remove the cell culture plate to be tested and equilibrate to room temperature (22°C-25°C). The luciferase reaction is sensitive to temperature changes. Detection reagents and experimental cell plates need to be equilibrated to room temperature, and the temperature should remain constant (±1°C) during testing.
4.Take out the lysis buffer, thaw completely, equilibrate to room temperature, and invert to mix thoroughly.
5.Take out the nano-luciferase substrate, centrifuge briefly to collect the contents at the bottom of the tube, and place on ice.
6.Prepare the nano-luciferase detection reagent: Determine the volume of reagent to prepare based on experimental needs. When preparing, add the nano-luciferase substrate at 200x to the lysis buffer and mix thoroughly to obtain the nano-luciferase detection reagent.
7.Add an equal volume of nano-luciferase detection reagent to each well of the experimental cell plate as the culture medium in the well. For example, add 100 μL of nano-luciferase detection reagent to 100 μL of culture medium. Shake the plate for 1 min to mix and incubate in the dark for 3-10 min.
8.Read the luminescence signal on a luminometer or multifunctional plate reader. The signal half-life is > 2 hr. Extremely high expression of nano-luciferase may accelerate substrate consumption, leading to a reduced signal half-life.
Guidelines
1. Mixing different batches is not recommended
2. Altering the dosage of detection reagents without rigorous validation is not recommended
3. For research use only
4. NanoLuc is a registered trademark of Promega Corporation
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Bioactivity
HEK293 cells were transfected with a plasmid containing the Nano-luciferase reporter gene. Forty-eight hours after transfection, the cells were seeded into a 96-well plate at 15,000 cells per well, with the cell number per well as indicated in the table. The cells were then assayed using Nano-Steady luciferase assay reagent equilibrated to room temperature, with culture medium containing no cells serving as the blank control. The values in the table represent the average of two replicate wells measured over a 20-minute incubation period.
