The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg 2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10 -20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.
The Luciferase Assay System was developed for reporter quantitation in mammalian cells. The Luciferase Assay System (Cat.# E1500), provided with Cell Culture Lysis Reagent (CCLR), also can be used for reporter quantitation in plant and bacterial cells; however, the Luciferase Assay System with Reporter Lysis Buffer (Cat.# E4030) is not suitable for these applications.
The Luciferase Reporter 1000 Assay System (Cat.# E4550) was designed to meet the needs of users who perform a large number of assays, particularly in 96-well plates. The system contains sufficient reagents to perform 1,000 luciferase assays (100µl per assay). For users working with transformed cells, a cell lysis buffer will be needed for sample preparation prior to luciferase measurement. The lysis buffer must be purchased separately.
Summary of Changes
The following changes were made to the 5/24 revision of this document:
1. Revised Section 3.D, Step 3.
2. Made minor text edits.
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