NLuc luciferase assay kit

NLuc luciferase is a genetically engineered small molecule enzyme (19.1kDa) that is a highly efficient bioluminescence reporter gene protein. It is about 150 times brighter than firefly (Photinus pyralis) or sea kidney luciferase, and can produce high-intensity, glow like luminescent signals using a new type of coelicin analogue (Furimazine). This luminescence reaction does not rely on ATP and achieves the highest detection sensitivity by suppressing the background luminescence signal.
The NLuc Reporter Gene Assay Kit provides a simple, single addition reagent with high sensitivity that can generate strong signals in the presence of NLuc luciferase.
Product components:

1. Self provided materials:
PBS； Multi channel firing; White opaque/black cell culture plate; Chemiluminescence analyzer or enzyme-linked immunosorbent assay (ELISA) reader.
2. Preparation before testing:
(1) Place NLuc detection substrate (50x) and NLuc detection substrate buffer at room temperature in advance.
(2) According to the actual usage, mix an appropriate amount of NLuc detection substrate (50x) with NLuc detection substrate buffer in a ratio of 1:50, and store at room temperature in the dark for future use. For example, if 10 mL of reaction solution is required, 0.2 mL of detection substrate needs to be added.
Note: It is recommended to prepare and use it immediately. The remaining reaction solution containing the new substrate is not recommended to be retained after the end of the experiment.
3. Operation method:
(1) Remove the cell culture plate from the incubator and let it sit for 5-15 minutes until it returns to room temperature.
Note: Use white opaque or black cell culture plates to reduce signal interference between wells.
(2) Add detection solution
Add a substrate containing reaction solution that has been equilibrated to room temperature to each cell well using a multi-channel pipette, with a sample volume equal to the volume of the cell culture medium, and mix well. For example, a 96 well plate usually contains 80-100 μ L of culture medium, followed by 80 μ L-100 μ L of detection solution; 384 well plates usually contain 20-30 μ L of culture medium, followed by 20-30 μ L of detection solution.
(3) Luminescence detection
After mixing, detect the activity of NLuc luciferase reporter gene in a chemiluminescence analyzer or enzyme-linked immunosorbent assay (ELISA) reader.
Note: To obtain the best detection results, please complete the test within 1 hour after adding the detection reagent.
4. Analyze data:
(1) Experimental design: According to different experimental purposes, a control group, an experimental group, and a blank control group should be set up in each culture plate. To ensure experimental accuracy, theoretically, each experimental group (including the control group) should subtract the luminescence measurement value of firefly luciferase from the blank control group.
① Blank control group:
Background F: Untransfected cells+reaction solution.
Note: The sample size of the blank control group must be the same as the experimental sample size, including the same culture medium/serum combination as the experimental sample, and adding the same detection reagent as Wan.
② Experimental group: Transfected cells were treated with experimental compounds (i.e. experimental group F).
③ Control group: Transfected cells without treatment were used to standardize the results (i.e. control group F).
(2) Calculation result:
Experimental group=Experimental group F - Background F.
Control group=Control group F - Background F.
Expression fold=experimental group/control group.

-Stored at 20 ℃, with a validity period of one year. NLuc detection substrate (50 x) is recommended to be packaged and stored at -80 ℃.

1. Enzymatic reactions are sensitive to temperature, and all reagents must be equilibrated to room temperature (20-25 ℃) before use for sample detection;
2. White or black 96 well plates are required for testing. If a regular transparent 96 well plate is used, there will be mutual interference between adjacent holes;
3. Testing instruments: Instruments that can detect chemiluminescence are suitable, but due to different instrument settings and sensitivities, the measured light signal values may also vary;
4. Single tube fluorescence analyzer should be used for measurement, and the time from mixing each sample with the measuring reagent to before measurement should be consistent;
5. For your own safety, please take effective protective measures before the experiment;