UA-Glo® Nano-luc Live Cell Assay System

The Nanoluc split enzyme technology divides the luciferase protein molecule into two different fragments, which are expressed separately. These two fragments have high affinity and can quickly and automatically form a complete enzyme molecule with luciferase activity in the same reaction system. The Nanoluc Live Cell Assay System provides reaction substrates that can enter cells. The two enzyme fragments of the reaction system are cloned into two expression vectors respectively, and each is expressed on the same molecule as two target proteins, serving as tags for the two target protein molecules. The luciferase activity of cells after co-transfection with the detection vectors can be used to study the expression levels, interactions, dynamic changes, etc., of the two target proteins in the cells.

Reaction buffer and substrate are separately mixed and filled in 10 ml or 100 ml brown bottles and 2 ml spiral tubes, with specifications as follows:

1. Plate the cells to be tested at an appropriate density in a 96-well or 384-well cell culture plate. The cells to be tested can be transiently transfected cells, stable cell lines, or endogenously expressing cell lines that express the target protein tagged with split luciferase fragments. It is recommended to use white plates.

2. Treat the cells according to the project requirements and continue culturing for an appropriate period.

3. Take out the detection buffer and equilibrate to room temperature. Centrifuge the substrate tube for 10 seconds to collect the contents at the bottom of the tube, and place it on an ice box.

4. Take out the cell culture plate to be tested and equilibrate to room temperature.

5. Prepare the detection reagent: It is recommended to add 50 µl of detection reagent to cells in a 96-well plate, or 10 µl of reagent to cells in a 384-well plate. Determine the amount of reagent to be prepared according to experimental needs. When preparing, add the substrate to the detection buffer and mix thoroughly. The substrate in the kit is 200x; before use, dilute it with buffer to a 1x detection working solution. Note to avoid light.

6. Aspirate the cell culture medium from the detection wells, add 50 µl of 1x detection working solution to each detection well of the 96-well plate, or 10 µl of reagent to each detection well of the 384-well plate, and gently shake for 15 seconds. For suspension cells, centrifuge the reaction plate, carefully aspirate the culture medium, and then add the 1x detection working solution.

7. Read the fluorescence signal on a fluorescence microplate reader. Save the data and perform data processing and analysis.

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

2.  Reagents from different batches are not recommended to be mixed for use.

3.  Subpackage storage: Subpackage and store the reaction reagents in accordance with the instructions to ensure the stability of the reagents.