UA-Glo® Nano luciferase Lytic Detection System

The Nano-luc Split Enzyme Technology divides NanoLuc® Luciferase into two high-affinity fragments: a large fragment and a small fragment. The small fragment of NanoLuc® is expressed as a tag for the target protein and can rapidly combine with the large fragment in the same reaction system to form a complete enzyme molecule with NanoLuc® activity. Detecting the NanoLuc® activity resulting from the binding of the large and small fragments can be used to study protein expression, regulation, degradation, and protein-protein interactions in cells.

U-Excell Bio's UA-Glo® Nano luciferase Lytic Detection System includes proprietary cell lysis buffer and NanoLuc® substrate. This system offers high detection sensitivity, low background, a wide detection range, and stable detection signals (with a half-life of approximately 3 hours), making it particularly suitable for high-throughput compound screening.

Users construct target protein expression vectors tagged with Nano luciferase small fragment and prepare cells expressing target proteins according to experimental requirements, including transient or stable exogenous expression, as well as endogenous expression cell lines generated via technologies such asCRISPR-Cas9 Knock-IN technology. The Nano luciferase large fragment must be provided by the user.

Seed cells at an appropriate density in white opaque96-well or384-well cell culture plates, transfect with plasmids expressing target protein tagged with Nano luciferase small fragment, or directly seed stable cell lines expressing the target protein.

1. Treat the cells as required by the experiment and continue culturing for an appropriate duration.

2. Equilibrate cell lysis buffer to room temperature (22–25°C). Luciferase reactions within detection reagents are temperature-sensitive. Reagents and test samples/plates must be equilibrated to room temperature (22–25°C), and temperature should be maintained constant (±1°C) throughout detection.

3. Take out substrate and Nano luciferase large fragment (user-supplied), centrifuge briefly for10 sec to collect liquid at the tube bottom, then place on ice.

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

5. Detection reagent preparation: Calculate required reagent volume according to experimental scale. For preparation, first add 200× substrate into cell lysis buffer and mix thoroughly, then add Nano luciferase large fragment (user-supplied). Optimal concentration of Nano luciferase large fragment requires optimization; its working concentration should be in excess relative to target protein concentration. The recommended stock concentration is 100×. Dilute it to 1× using the above 1× substrate-containing lysis buffer prior to use and mix well.

6. Add detection reagent in equal volume to the culture medium in each well. For example, add 100 μL detection reagent to 100 μL culture medium per well. Shake the plate for 3 min, then incubate in the dark for an additional 10 min to allow cell lysis.

7. Measure luminescence signal with a multi-mode microplate reader.

1. This product is for research use only.

2. Mixing reagents from different batches is not recommended.

3. Modification of detection reagent dosage without rigorous validation is not recommended.

4. Store detection reagents as instructed to guarantee reagent stability.