Protocol II (Injector-equipped luminometers) (E3309)
Protocol
- Thaw BioLux Cypridina Luciferase Assay Buffer (1X) completely to room temperature (protect from light) and mix well before use.
- Prepare the CLuc assay solution (e.g. for 100 samples add 50 μl of the reconstituted substrate (100X solution) to 5 ml of BioLux Cypridina Luciferase Assay Buffer).
Note: Be sure to prepare enough CLuc assay solution as needed for all samples as well as for priming the injector as suggested by the manufacturer. - Mix well by inverting the tube several times (do not vortex).
- Incubate at room temperature for 30 minutes (protect from light).
- Set the luminometer with the following parameters: 50 μl injection, 1–2 seconds of delay and 2–10 seconds of integration.
- Pipet samples *(5–20 μl per well) into a 96-well white (opaque) or black plate.
- Prime the injector with the CLuc assay solution and proceed with the measurement.
* Approximately 90% of Cypridina Luciferase is secreted into the culture medium after transfection and thus, the CLuc activity is typically assayed in the supernatant (i.e. culture medium of CLuc-transfected cells). However, as long as the cells are alive, ~10% of CLuc is present inside the cell and therefore, the CLuc activity can also be assayed in the cell lysate (Figure 7 on the product page). We recommend that the cell lysates be prepared by using Luciferase Cell Lysis Buffer (#B3321), since this lysis buffer is designed to be compatible with Cypridina, Gaussia, Renilla, Firefly Luciferase and β-gal activity assays.
