Luciferases

Firefly Luciferase

The most versatile and common reporter gene is the luciferase of the North American firefly photinus pyralis. The protein requires no posttranslational modification for enzyme activity. It is not even toxic in high concentration (in vivo) and can be used in pro- and eukaryotic cells.

Amplite™ Luciferase Reporter Gene Assay

Amplite™ Luciferase Reporter Gene Assay Kits use a proprietary DTT-free formulation to quantify luciferase activity in live cells and cell extracts. The assay is based on firefly luciferase, a monomeric 61 kD enzyme that catalyses a two-step oxidation of luciferin, which yields light at 560 nm. Our formulation generates a luminescent product that gives strong luminescence upon interaction with luciferase. Kits include all the essential components with an optimized “mix-and-read” assay protocol that is compatible with HTS liquid handling instruments. They have high sensitivity and can be used for the assays that require low detection limit. The kits have a fast, simple, and homogeneous bioluminescence assay for studying gene regulation and function. The assay is compatible with the use of standard cell growth media.

Figure 1. Luciferase dose response was measured with Amplite™ Luciferase Reporter Gene Assay Kit in a white 96-well plate with a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 0.1pg/well luciferase with 20 minutes to 5 hours incubation without losing signal intensity. The integration time was 1 second. The half life is more than 4 hours.

Guassia Luciferase

In recent years, there has been an increase in the use of other luciferases, such as Gaussia luciferase, since these reporters are smaller and do not require the presence of ATP. Gaussia luciferase, which is a 20 kD protein, catalyzes coelenterazine oxidation by oxygen to produce light. The bioluminescent enzyme derived from the marine copepod Gaussia princeps is efficiently secreted from mammalian cells upon expression.

Amplite™ Gaussia Luciferase Reporter Gene Assay

Amplite™ Gaussia Luciferase Reporter Gene Assay Kits use a proprietary luminogenic formulation to quantify luciferase activity in cell medium. The formulation generates a luminescent product that gives strong luminescence upon interaction with Gaussia luciferase. The kits provide all the essential components that are compatible with HTS liquid handling instruments. They have high sensitivity and can be performed in convenient 96-well and 384-well microtiter-plate formats. The “glow-type” signal with a half-life of one hour provides a consistent signal across large number of assay plates. The assay is compatible with standard cell growth media.

Figure 2. Sectreated Gaussia Luciferase culture medium was measured with Amplite™ Gaussia Luciferase Reporter Gene Assay Kit (blue line) and a commercially available Gaussia Luciferase Assay Kit (red line) in a white 96-well plate with a NOVOstar plate reader (BMG Labtech).

Renilla Luciferase

Renilla luciferase is a 36 kDa protein isolated from the sea pansy (Renilla reniformis). Compared to firefly luciferase, Renilla luciferase enzymes differ in their substrate and cofactor requirements. Renilla luciferase uses coelenterazine in the presence of oxygen to produce a blue light of 480 nm. Similar to firefly luciferase, Renilla luciferase can be used in dual-reporter assays due to their differences in substrate requirements and light output.

Amplite™ Renilla Luciferase Reporter Gene Assay

Amplite™ Renilla Luciferase Reporter Gene Assay Kits offer a fast and sensitive method to detect Renilla luciferase activity in cell-based assays using a proprietary luminogenic formulation. Upon interatcion with Renilla luciferase this formulation generates a product with strong luminescence. The “glow-type” signal with a half-life of one hour provides a consistent signal across large number of assay plates. The assay is compatible with standard cell growth media. The kits enable the measurement of primary expression or gene expression with wild type and synthetic hRluc genes. Each kit includes all the essential components and can be performed in a convenient 96-well and 384-well microtiter-plate format.

Figure 3. Renilla Luciferase was measured with Amplite™ Renilla Luciferase Reporter Gene Assay Kit in a white 96-well plate with a NOVOstar plate reader (BMG Labtech).

Table 1. Specific Luciferase Specificity, Cofactor Requirements and Physical Characteristics.