Luciferase Assay

Method: Assay of luciferase activity is based upon the measurement of light produced. Although units of activity have been proposed for the purified enzyme (see discussion) currently, no units have been assigned to the Worthington preparations.

Reagents

• 0.01% Mercaptoethanol
• 0.1 M Sodium phosphate buffer, pH 6.8
• 13 mM Nicotinamide adenine dinucleotide, reduced form (NADH)
• 0.42 mM Flavin mononucleotide (FMN)
• 0.1% Decaldehyde in methanol

Enzyme

Dissolve at 10 mg/ml in reagent grade water.

Procedure

A spectrophotometer capable of a percent transmission readout is used for the determination. With the light source off and the UV detector in position, adjust the readout to 0% transmission.

Add to each cuvette:

0.01% Mercaptoethanol	0.1 ml
0.1 M Sodium phosphate buffer, pH 6.8	1.3 ml
13 mM NADH	0.2 ml
0.42 mM FMN	0.2 ml
Enzyme solution	0.2 ml

Mix contents well and at zero time add 0.05 ml of 0.1% decaldehyde solution. Read the maximum transmission as a measure of the light produced. Addition of excess enzyme will enable the light produced to be visualized by the dark adapted eye.