UA-Glo® Nano luciferase Live Cell Assay System

The NanoLuc Split Enzyme Technology divides NanoLuc into two distinct fragments: a large fragment and a small fragment. Individually, these fragments lack enzymatic activity, but when combined, they restore NanoLuc activity. The two fragments can have different combinations of high or low affinity. High-affinity fragments can rapidly combine in the same reaction system to form enzymatically active NanoLuc, while low-affinity fragments can rely on the binding of their tagged target proteins to become enzymatically active NanoLuc. Detecting the NanoLuc activity resulting from the binding of these fragments can be used in research areas such as cellular protein expression, regulation, degradation, and protein-protein interactions.

The UA-Glo NanoLuc Intracellular Detection Kit includes Youai Biotech's proprietary cell-permeable NanoLuc substrate and buffer, enabling the detection of NanoLuc activity resulting from fragment binding in live cells without cell lysis, as well as the detection of NanoLuc expression in live cells. This kit offers high sensitivity, low background, a broad detection range, and stable detection signals (with a half-life of approximately 2 hours), making it particularly suitable for high-throughput compound screening.

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1. Seed experimental cells at an appropriate density in 96-well or 384-well cell culture plates. The cells can be transiently transfected cells, stable cell lines, or endogenously expressing cell lines that separately express two target proteins tagged with the large and small fragments of NanoLuc luciferase. White plates with clear bottoms are recommended.

2. Treat the cells according to experimental requirements and continue culturing for an appropriate duration.

3. Remove the detection buffer and equilibrate to room temperature (22℃-25℃). The luciferase reaction is sensitive to temperature changes. Reagents and assay plates must be equilibrated to room temperature, and the temperature should remain constant (±1℃)

4. Centrifuge the NanoLuc substrate tube to collect the contents at the bottom, and place on ice.

5. Remove the experimental cell culture plate and equilibrate to room temperature.

6. Determine the volume of detection reagent based on experimental requirements. It is recommended to add 96-well plate per well 50 µL of detection reagent, 384-well plate per well 10 µL of detection reagent.

7. Prepare the detection reagent: The NanoLuc substrate is supplied as a 200x concentrate. Dilute to 1x with buffer before use. Mix thoroughly. Protect from light.

8. Select one of the following detection methods based on experimental needs:

   1. After discarding the culture medium from the experimental cell plate, directly add 96-well plate per well 50µL of detection reagent(384-well plate add 10µL), this method provides the highest signal intensity and lowest detection background.

   2. After discarding the culture medium, add 96-well plate per well 100µL of serum-free medium (e.g., Opti-MEM (384-well plate add 20µL), then add 50µL of detection reagent(384-well plate add 10µL). This method helps maintain better cell viability.

   3. Without discarding the culture medium, directly add 96-well plate 50µL of detection reagent(384-well plate add 10µL): This method is suitable for HTS experiments. FBS in the culture medium can affect signal intensity and increase background. Cells can be seeded in low-serum medium, such as FBS to minimize the impact of 2% FBS to seed experimental cells and reduce the impact of FBS. The optimal FBS concentration may require optimization.

9. a: For method a, incubate directly in the dark for 10min. For methods b and c, gently shake the plate to mix, then incubate in the dark for 10min

10. Read the luminescence signal on a multi-mode microplate reader.