The Luciferase Reporter Gene Assay contains all substrate components for the enzymatic determination of firefly luciferase activity, as well as a lysis buffer for the mild and rapid extraction of luciferase from eukaryotic cells. In the first step, transfected cells are lysed by the addition of lysis buffer. Subsequently, the cell extract is transferred to a microplate or tube, depending on the type of equipment used. The enzymatic reaction is then started by adding the mixture of reaction buffer and luciferase substrate (luciferase assay reagent), which contains all of the components required for starting the chemiluminescent reaction. The luciferase assay reagent may be added manually or by automated injector. Photon emission is quantitated using a luminometer or alternative equipment such as a scintillation counter.
The luciferase assay is much more sensitive (100- to 1000-fold) than the standard isotopic CAT (chloramphenicol acetyltransferase) assay. The Luciferase Reporter Gene Assay is not suitable for the determination of luciferases of bacterial origin.
- Reaction Buffer
- Luciferase Substrate
- Lysis Buffer
Optimized to detect the widely used monitoring gene that encodes firefly Photinus pyralis luciferase
The Luciferase Reporter Gene Assay is used to quantitatively measure the expression of firefly luciferase in eukaryotic cells or bacteria transfected with a vector that encodes firefly Photinus pyralis(EC 126.96.36.199) luciferase. The Luciferase Reporter Gene Assay can be used in manual or automated luminometers in a microplate or tube format, as well as in scintillation counters or photographic films. In order to achieve maximum sensitivity, the use of a luminometer that operates with ultra-fast photon counters is recommended (e.g., EG&G Berthold luminometers).
Features and Benefits
- Linear light emission: Optimized luminescent reaction produces a linear glowing light instead of an ultra-short flash
- Extremely sensitive: More sensitive than the linear light-emitting range of firefly luciferase enzyme
- Detection hardware: Can be performed in all automated or manual microplate- or tube-format luminometers or other counters
- Faster: Faster than any other reporter-gene assay
- Compatible: The optimized formulation of the lysis buffer allows evaluation of the sample using the nonradioactive CAT ELISA or hGH ELISA in cotransfection experiments.
- Influence of coenzyme A (CoA) on luciferase light emission kinetics. Roche′s Luciferase Reporter Gene Assay, which contains CoA (upper curve), in comparison to a standard luciferase assay without CoA (lower curve). In the reaction with no CoA added, more than 50% of the total light emission is produced within the first 10 seconds. The use of CoA leads to linear light production over 20 - 30 seconds and decays with a half-life of approximately 5 minutes.
- Assay time: <30 minutes
- Detection range: Light emission is linear within a range of approximately 10 fg to 10 ng (1 pg/ml to 1 μg/ml based on a sample volume of 10 μl). The upper limit of the detection range depends on the measuring device used.
- Sample material: Extracts from eukaryotic cells or bacteria, expressing firefly luciferase from Photinus pyralis (e.g., as a reporter protein).
- Sensitivity: 5 fg (500 fg/ml based on a sample volume of 10 μl) luciferase.
- Simple to perform: Easy to handle
- Function tested
1 kit containing 3 components
1 set containing 3 components
Activator: – phosphate
Storage conditions (working solution): The Luciferase assay reagent is stable for 12 months at -60 °C or below, for
1 month at -15 to -25 °C if stored in aliquots, or for 1 week at 2 to 8 °C. Avoid repeated freezing and thawing. Store protected from light, as luciferin is oxidized when exposed to light.
The Lysis Buffer is stable until the expiry date given on the kit if stored in aliquots at -15 to -25 °C.
For life science research only. Not for use in diagnostic procedures.
Gene expression in transfected eukaryotic or prokaryotic cells is generally studied by linking a promoter sequence to an easily detectable "reporter" gene, such as that which encodes for luciferase. Firefly luciferase may be used to monitor promoter activity in vitro in cultured animal cells, bacteria, insects, plants, yeast, and viruses.
The chemiluminescent assay for firefly Luciferase activity is an extremely sensitive, rapid, easy-to-handle, and non-isotopic alternative to other reporter-gene assay systems. The luciferase assay is much more sensitive (100- to 1000-fold) than the standard isotopic CAT assay. The detectable linear range of firefly luciferase is approximately 10 fg/ml to 1 μg/ml (10-16M - 10-8M). Firefly luciferase from Photinus pyralis (EC 188.8.131.52) catalyzes the adenosine triphosphate (ATP)-dependent oxidative decarboxylation of luciferin, producing light emission at a wavelength of 562 nm. The quantum yield 0.88 of the reaction is the highest known for chemiluminescence reactions. The kinetics of luciferase-catalyzed light emission are characterized by a transient flash that peaks after 0.3 - 0.5 seconds, then rapidly decays. The main flash reaction is finished within approximately 20 seconds. Thus, classical luciferase determination requires rapid mixing and starting of the light-emission measurement.
Production of constantly glowing light emission over a period of several minutes without a significant decrease in peak height can be achieved by adding Coenzyme A (CoA) to the Luciferase Reporter Gene Assay (see below). The Luciferase Reporter Gene Assay thus allows precise quantification of luciferase activity while avoiding an ultra-short flash.