Standard Assay Protocol I (Luminometers without injectors) (E3300)
- Prepare the GLuc assay solution (e.g. 100 samples) by adding 50 μl of BioLux GLuc Substrate to 5 ml of BioLux GLuc Assay Buffer immediately before performing the assay.
- Mix well by inverting the tube several times (Do not vortex).
- Set the luminometer for 2–10 seconds of integration.
- Pipet samples* (5–20 μl per well) into a 96-well white (opaque) or black plate, or a luminometer tube.
- Add the GLuc assay solution (50 μl) to a sample (i.e. Add the assay solution to only one sample at a time) and promptly measure the luminescence.
- Repeat Step 5 for all samples.
* Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate. We recommend that the cell lysates be prepared by using Luciferase Cell Lysis Buffer
(NEB #B3321), since this lysis buffer is designed to be compatible with Cypridina, Gaussia, Renilla, Firefly luciferase and β-galactosidase.