Luciferase Reporter Assay Kit

Luciferase Reporter Assay Kit

Cat. No. abs-81                                                                                                                                           Size: 50 Assays

â… . Introduction:

The Luciferase Assay Kit provides a rapid, sensitive, and quantitative measurement of the activity of the reporter enzyme American firefly (Photinus pyralis) luciferase in cultured mammalian cells. Firefly luciferase has been used as a sensitive reporter for studying gene regulation and function. Firefly luciferase catalyzes the oxidative carboxylation of luciferin, a reaction with the highest efficiency of any known bioluminescence reaction. The light emission from the reaction can be recorded using a luminometer. GeneDirex’s Luciferase Reporter Assay Kit provides a simple means for detecting luciferase activity in transfected eukaryotic cells. For measurement of expressed luciferase in vitro, luciferase is first extracted from transfected cells through cell lysis. Then CoA, ATP, Mg2+ and buffer are added to the lysate (Inclusion of CoA yields a nearly constant light emission rather than typical flash Kinetics). The luminescent reaction is then triggered by an injection of luciferin, and the emitted light is recorded by a luminometer. By providing CoA, along with ATP, Mg2+ in an optimized buffer solution, GeneDirex Luciferase Reporter Assay Kit ensure maximal sensitivity, consistent light output, as well as convenience and consistency when working with multiple samples.

â…¡. Kit Contents:

â…¢. General Consideration and Reagent Preparation:

l  Store Luciferase Reporter Assay kit at -70oC.

l  Ensure that all reagents have reached room temperature before performing assays.

l  The following protocol is designed for using with adherent cultures growing at 35-mm tissue culture plates. If you are using plates of different size, adjust the volume proportionally. Read entire protocol before starting experiments.

â…£. Assay Protocol:

A. Preparation of Cell Lysate

1. Remove media from cell culture plates and rinse once with PBS, being careful to not dislodge attach cells.

2. Dilute Cell Lysis Buffer 1 vol. to 4 vol. H₂O freshly before use.

3. Add enough 1X Cell Lysis Buffer to cover the cells (e.g. 400 ul/60mm culture dish, 900 ul/100mm culture dish, 50 ul/96-well plate).

4. Rock culture dishes several times to ensure complete coverage of the cells with Cell Lysis Buffer. Scrape attached cells from the dish. Transfer cells and all liquid to a microcentrifuge tube. Place the tube on ice.

5. Vortex the microcentrifuge tube 10-15 seconds, then centrifuge at 12,000 rpm. Transfer the supernatant to a new tube.

6. Store the supernatant/cell lysate at -70℃

B. Luciferase Assay:

1. Program the luminometer for the appropriate delay and measurement times.

2. Place 35 μl cell extract into an assay microplate well. Be sure to use the same volume.                                 

Note: The amount of extract required may vary depending on the luciferase expressi on level and the instrumentation used; the amount used should be adjusted to keep the signal within the linear range of the assay.

3. Add 10 ul ATP (100X) into 1ml Luciferase Assay Buffer.

4. Add 40 μl Luciferase Assay Buffer (with ATP) into each well.

5. Within 10 minutes, add 20 μl/well Luciferase Assay Substrate. Read the signal immediately using a  luminometer. Note: The time between adding substrate B and reading signal should be as short as possible (1-2 seconds) and consistent from sample to sample.