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

  1. Prepare the GLuc assay solution (e.g. 100 samples) by adding 50 µl of BioLux GLuc Substrate and 800 µl of BioLux GLuc Stabilizer to 5 ml of BioLux GLuc Assay Buffer (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. Incubate at room temperature for 25 minutes (protect from light in a tightly capped tube/bottle) before adding to the sample.
  4. Set the luminometer with following parameters: 50 µl of injection, 35-40 seconds of delay (refer to Notes), & 2-10 seconds of integration.
  5. Pipet samples* (5-20 µl per well) into a 96-well plate (opaque, white or black) or a tube.
  6. Prime the injector with the 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 cell.  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.