Application

Firefly luciferase is used extensively in molecular and cell biology, in particular for the efficient detection and quantitation of ATP and as a reporter for genetic function.

Biochem/physiol Actions

Firefly luciferase is a 62 kDa protein that catalyzes the production of light. The enzyme requires ATP, molecular oxygen, and the heterocyclic compound, firefly luciferin, to generate light in a two-step process. The light producing reaction is initiated by luciferin activation (adenylation of its carboxylate group) and proceeds in the presence of molecular oxygen to yield a photon of yellow-green light.

Physical form

Supplied as a lyophilized powder containing HEPES, pH 7.5, NaCl, MgCl₂, EDTA, DTT and a carbohydrate stabilizer.

This product is a recombinant luciferase (62 kDa) from Photinus pyralis (American firefly) produced from the luc gene expressed in E. coli.

Preparation Note

To obtain maximal solubility it is important to reconstitute the enzyme at a high salt concentration, such as 1 M Tris buffer with any counter ion at pH 7-8. The enzyme can be prepared at a concentration of up to 5 mg protein/ml. Do not vortex and avoid agitation.After reconstitution, the enzyme solutions can kept at 4-8 °C for up to 2 days or frozen in working aliquots at -20°C for at least one month. Repeated freezing and thawing is not recommended.

Unit Definition

One luciferase enzyme unit will produce one Relative Light Unit (RLU) at 20-25 °C over a 10 second period, measured in 100 µl assay mixture containing 40 pmol ATP and 15 nmol luciferin in Tris-glycine buffer, pH 7.6, using a GloMax 20/20 Luminometer.Unit Definition Conversion Factor: There are approximately 9000 Relative Light Units (RLU) per one traditional Light Unit that uses a peak height equivalent to 0.02 µCi of ¹⁴C in a PPO/POPOP cocktail.