Qin Hao Jan 2006; Court Feb 2008
Firefly Luciferase Activity Assay
Introduction
D-luciferin is the substrate for firefly luciferase (Photinus pyralis). Luciferase catalyzes
the oxidation of luciferin to oxyluciferin in the presence of ATP and magnesium,
resulting in bioluminescense.
The luciferin/luciferase system is used as a very sensitive reporter assay for gene
expression, a plasmid containing firefly luciferase can be used as a co-transfector to
compare the efficiency of transfection.
Sample Reporter Reagent Formulation
Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and
15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in
polypropylene or glass.
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D-luciferin sodium salt (Gold Bio Technology, Inc. Catalog No. LUCNA-100)
FW=302.3
ATP disodium salt (Sigma, Catalog No. A7699-1g) FW=551.1
MgSO47H2O FW=246.5
1 M HEPES buffer
For example: If want to prepare 50 ml luciferin substrate reagent, weigh 15.1 mg Dluciferin salt, 82.7 mg ATP salt, 185 mg MgSO47H2O, add to 50 ml polypropylene
tube, then add 1.5 ml 1 M HEPES buffer, 48.5 ml ATP free water, vortex, make sure all
compounds dissolve completely.
Sample Reporter Assay Protocol
1. Warm luciferin substrate reagent to room temperature before starting.
2. Sonicate cells.
3. Pipet 250ul substrate reagent to the tube of Lumat LB 9501(in M&V 302),
measure background, then add 2.5ul cell sample, mix, measure again.
(Note: the volume of substrate reagent and cell sample can be changed)
Qin Hao Jan 2006; Court Feb 2008
Tips on Setting Up and Running the Lumat LB 9501 (by Ben Harwood)
Setting up Luminometer for Luciferase Assay:
1. Main Menu > Other > Inst Parameters:
2. Injection time > Default
3. Back Press Inject 1> Default
4. Back Press Inject 2> Default
5. Enter Delay 1st/ 2nd Inject > 5-7s (critical)
6. Enter Delay last Inject > Default
7. >save changes.
8. Then run luciferase assay.
When Running Samples:
1. Put blank sample with no luciferase activity in machine and start measurement.
2. Once background is measured and screen says “injecting” open up arm and switch
in tube with luciferase activity.
3. You must switch the tube while it is “injecting” since before will not allow for a
measurement.
4. And during the measurement if arm is open, measurement will also fail.