Manual - New England Biolabs

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4. Add RSL1 ligand to a final concentration of 500 nM to three wells and an equivalent
amount of DMSO to the remaining wells as a control. RSL1 can be added to
cells by one of two methods: 1) RSL1, diluted in DMSO, can be added directly to
each well; or 2) Culture medium can be replaced with fresh medium containing
500 nM RSL1.
Notes: a) DMSO concentration should not exceed 0.1 % (v/v) per well. b) RSL1 is not
soluble in culture media at concentrations higher than 10 µM. c) Level of expression
can be adjusted by varying the final RSL1 concentration.
5. Collect at least 20 µl of cell culture supernatant from each well 4–24 hours postinduction
for luciferase assay. Samples may be stored at –20°C without loss of
activity.
Gaussia luciferase assay:
i. Prepare assay working solution by diluting GLuc Substrate 100-fold in GLuc
Assay Buffer. 50 µl of working solution is required for each assay.
ii. Pipet 20 µl of each culture supernatant into a microtiter plate well or sample tube
as appropriate for luminometer being used.
iii. Add 50 µl of assay working solution to the sample and promptly measure the
luminescence. Integrate for 5–10 seconds.