FAQspeak

How do I prepare a firefly luciferase standard curve?

To prepare a luciferase standard curve, use QuantiLum® Recombinant Luciferase (Cat.# E1701) diluted in a compatible buffer containing 1mg/ml of BSA as a carrier protein. To choose a buffer compatible with the luciferase assay system to be used, please consult the lysis buffer compatibility table.

Dilute the QuantiLum® Recombinant Luciferase to create a stock solution of 5 × 10–12 moles/µl, as shown below. The BioMath Calculator can be used to convert the protein concentration of the 61kDa QuantiLum® luciferase to a molar concentration. For example, if the protein concentration of the QuantiLum® luciferase is 15.5mg/ml, this would represent a molar concentration of 2.5 × 10–10 mol/µl. Dilute this stock 1:50 to a concentration of 5 × 10–12 mol/µl (tube 1), then do tenfold dilutions to 5 × 10–21 mol/µl. Mix well after each dilution.

Tube QuantiLum® Recombinant Luciferase Volume Dilution Buffer Volume Final Concentration (moles/µl)
1 2μl of QuantiLum® luciferase stock
(Cat.# E1701)		 98µl 5 × 10–12
2 10μl from tube 1 dilution 90µl 5 × 10–13
3 10μl from tube 2 dilution 90µl 5 × 10–14
4 10μl from tube 3 dilution 90µl 5 × 10–15
5 10μl from tube 4 dilution 90µl 5 × 10–16
6 10μl from tube 5 dilution 90µl 5 × 10–17
7 10μl from tube 6 dilution 90µl 5 × 10–18
8 10μl from tube 7 dilution 90µl 5 × 10–19
9 10μl from tube 8 dilution 90µl 5 × 10–20
10 10μl from tube 9 dilution 90µl 5 × 10–21

To perform the luciferase assay, add 20µl of the diluted enzyme (tubes 3–10) to 100µl of the Luciferase Assay Reagent. If using a “glow-type” luciferase reagent (e.g., Bright-Glo™, Steady-Glo® or ONE-Glo™ Reagent), use equal volumes of reagent and diluted enzyme.
Note: The limits of detection will depend upon the reagent and the luminometer used.