Standard Assay Protocol II (Injector-equipped luminometers) (E3300)

Protocol

1. 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. (Be sure to prepare enough assay solution as needed for all samples as well as for priming a particular luminometer as recommended by the manufacturer).
2. Mix well by inverting the tube several times (Do not vortex).
3. Set the luminometer with the following parameters: 50 μl of injection volume and 2–10 seconds of signal integration.
4. Pipet samples* (5–20 μl per well) into a 96-well white (opaque) or black plate, or a luminometer tube.
5. Prime the injector with the GLuc assay solution and proceed with the measurement. 
   
   * 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-gal activity assays.