UA-Glo® Nano luciferase split Extracellular Assay System

The NanoLuciferase split enzyme technology divides the luciferase protein molecule into two distinct fragments, which are expressed separately. These fragments exhibit high affinity and can rapidly self-assemble into a complete, enzymatically active luciferase molecule within the same reaction system. Detection of the split NanoLuciferase activity can be utilized to study the expression levels, interactions, and dynamic changes of target proteins in cells.
The extracellular detection kit for split NanoLuciferase provides a non-lytic cell buffer and reaction substrate, while the small fragment of the split luciferase is cloned into an expression vector and co-expressed with the target protein as a tag. This target protein is either secreted or located on the extracellular side of the cell membrane. The expression vector carrying the small split luciferase fragment-tagged target protein must be constructed, prepared, and transfected into cells by the user.

The buffer and substrate are separately filled into 10 ml or 100 ml brown bottles and 2 ml spiral tubes, with the specifications as follows:

1. Seed the cells to be tested at an appropriate density in 96-well or 384-well cell culture plates. The cells to be tested can be transiently transfected cells expressing the target protein with a split luciferase small fragment tag, stable cell lines, or endogenously expressing cell lines. White plates are recommended.

2. Perform relevant treatments on the cells according to project requirements and continue culturing for an appropriate duration.

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

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

5. Prepare 1x assay working solution: Determine the volume of reagents to be prepared based on experimental needs. During preparation, add the required substrate and split enzyme large fragment (user-provided, optimal concentration needs optimization) to the buffer in the specified ratio, and mix thoroughly. The substrate in the kit is 200x, and it is recommended to prepare the split enzyme large fragment at a concentration of 100x. Dilute with buffer to 1x assay working solution before use. Protect from light.

6. Add an equal volume of 1x assay working solution to the test wells, e.g., add 100 μL of 1x assay working solution to 100 μL of medium. Shake the plate for 3 minutes and place it in the dark to continue lysing for 10 minutes.

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

1.         Reagent dosage: It is not recommended to arbitrarily change the amount of reaction reagents without rigorous validation.

2.         Reagents from different batches should not be mixed.

3.         Aliquot storage: Store reaction reagents according to the instructions to ensure reagent stability.