Reporter Gene Assays Support—Getting Started

The Thermo Scientific™ Pierce™ Luciferase reporter genes are human codon optimized and offered in a plasmid vector that is optimized for mammalian expression. See the table below for their key features:

• The Gaussia, Gaussia-Dura (a mutant form of the Gaussia Luciferase gene that confers better bioluminescent signal stability than native Luciferase) and Cypridina Luciferase proteins are secreted into the cell culture media, allowing for live cell monitoring of reporter activity. The light signal produced by these three secreted Luciferases is considerably greater than the signal from either native Firefly or Renilla Luciferases. Although greater than 85% of the Gaussia, Gaussia Dura and Cypridina Luciferase protein is secreted, the remaining intracellular portion can be robustly detected in lysate, facilitating multiplexing with other intracellular Luciferases. 
• The Green Renilla Luciferase is an intracellular protein with a greater serum stability as well as higher light output than native Renilla and Firefly Luciferases. Consequently, Green Renilla reporter assays are more sensitive than assays employing native Renilla Luciferase. 
• The Red Firefly Luciferase is an intracellular protein with an emission peak shifted into the red spectrum compared to the native Firefly Luciferase, which allows multiplexing with Gaussia, Cypridina and Green Renilla Luciferases.

We offer the following luciferase assays (each available in Glow and Flash formats) that provide a highly sensitive assay for transcriptional activity of regulatory elements in mammalian cell culture media and whole cell lysate:

• Thermo Scientific™ Pierce™ Gaussia Luciferase assays provide a highly sensitive system for detecting intracellular and secreted luciferase activity from promoter or pathway activation in mammalian cell culture experiments. Gaussia luciferase has greater protein stability and signal brightness than native Firefly or Renilla luciferase. The bioluminescent signal produced by Gaussia luciferase results from the oxidation of coelenterazine. This reaction does not require adenosine triphosphate (ATP) or other cofactors. The light output correlates with the amount of Gaussia protein expressed, which is proportional to the activity of the promoter for Gaussia expression.
  Note:Gaussia luciferase is a ~22 kDa protein.
• Thermo Scientific™ Pierce™ Renilla Luciferase assays provide a highly sensitive system for detecting intracellular luciferase activity from promoter or pathway activation in mammalian cell culture experiments. The bioluminescent signal produced by green Renilla luciferase results from the oxidation of coelenterazine. This reaction does not require adenosine triphosphate (ATP) or other cofactors. The light output correlates with the amount of green Renilla protein expressed, which is proportional to the activity of the promoter for green Renilla expression.
  Note: Unlike Gaussia and Cypridina, Green Renilla Luciferase can only be assayed in cell lysatesas it is not secreted into the cell culture media. Green Renilla luciferase is a ~36 kDa protein. 
• Thermo Scientific™ Pierce™ Cypridina Luciferase assays provide a highly sensitive system for detecting intracellular and secreted luciferase activity from promoter or pathway activation in mammalian cell culture experiments. The Cypridina luciferase protein is highly secreted into the cell culture media, allowing for live cell monitoring of reporter activity. Cypridina luciferase has greater protein stability and signal brightness than native firefly or Renilla luciferase. The bioluminescent signal produced by Cypridina luciferase results from the oxidation of vargulin. This reaction does not require adenosine triphosphate (ATP) or other cofactors. The light output correlates with the amount of Cypridina protein expressed, which is proportional to the activity of the promoter for Cypridina expression. 
  Note:Cypridina luciferase is a 61kDa protein.
• Thermo Scientific™ Pierce™ Firefly Luciferase Assays provide a highly sensitive system for detecting intracellular luciferase activity from promoter or pathway activation in mammalian cell culture experiments. The bioluminescent signal produced by firefly luciferase results from the oxidation of D-Luciferin. The light output correlates with the amount of firefly protein expressed, which is proportional to the activity of the promoter for firefly expression. 
  Note: Unlike Gaussia and Cypridina, Firefly Luciferase can only be assayed in cell lysates as it is not secreted into the cell culture media. 
  Note: Firefly luciferase is a ~60 kDa protein produced in nature by several species of the Lampyridae family of beetles which includes the genera Photinus and Luciola.

See the table below for the Luciferase Glow assay and Luciferase Flash assay kit options we offer:

Gaussia and Cypridina Luciferase assayscan be tested on both media and cell lysate sample types. Renilla and Firefly Luciferase assays can only be used on cell lysate as a sample type. 

We always recommend the luciferases to be read without filters for single luciferase assays. Use of filters reduces the amount of signal captured that may lead to decrease in sensitivity. Filters are required only for dual-spectral assays.

The earliest detectable signal will depend on the strength of the promoter. In general, with a strong promoter (such as CMV), significant signal over background can be seen in as little as 20 minutes. However, with a weaker inducible promoter, significant signal over background may take 1-2 hours after induction.

Gaussia, Gaussia-Dura, and Cypridina Luciferase are stable in the culture media for greater than 24 hours. Generally, after transfection of CMV driven constructs, we see ever increasing secreted signal between 24 and 72 hours.

The below table provides a guideline based on sample type and luciferase of choice. A luminometer with injectors is recommended for Flash assays.

The three luciferase reporters, Gaussia, Cypridina and Renilla Luciferase are paired with Firefly Luciferase as a normalization control. For example, Gaussia Luciferase acts as an experimental reporter with constitutively active Red Firefly Luciferase as a normalization control. This reporter-and-control combination enables simultaneous monitoring of experimental reporter and control luciferase activities in a single-read assay without the need for two-step addition of substrate reagents or quenching. The assay working solution contains substrates for both luciferases, and the reactions occur simultaneously with flash-type kinetics. The resulting luminescent signals are spectrally resolvable using filters. In a single sample, researchers can assay transcriptional activity of regulatory elements, signal transduction pathways, and effects of activators or inhibitors.

No, our dual spectral Luciferase assay kits are not useable with Promega’s Firefly/Renilla reporter vectors. This is because our Firefly Luciferase emits in red range and our Renilla Luciferase emits in green range, while Promega’s Firefly Luciferase emits in green range and their Renilla Luciferase emits in blue range. 

Promega offers a number of luciferase assays, but due to Promega’s licenses for their products, we cannot recommend the use of any of our reagents with their vectors or our vectors with their reagents. 

TurboLuc ™ Luciferase One-Step Glow Assay Kit measures activity in mammalian cells with a single reagent-addition step, making it ideal for high-throughput screening (HTS) applications. Several features of the TurboLuc ™ Luciferase One-Step Glow Assay Kit make it suitable for luminometers without injectors and for HTS applications. The convenient, one-step, homogenous protocol minimizes handling steps to support the use of automation. Simply combine the supplied substrate solution and assay buffer to make a single working solution, and then add it to microplate containing the transfected, treated cells. The stable signal output (glow enzyme kinetics) of this system provides flexibility with regard to lead time before read. When used with Thermo Scientific™ TurboLuc™16 Luciferase vectors, the TurboLuc ™ Luciferase One-Step Glow Assay Kit provides a highly sensitive bioluminescent reporter assay system for the detection of promoter or pathway activity.

There are three vectors available for each expressed luciferase; pMCS, pCMV, and pTK. We recommend using pMCS to measure the activity of the reporter, and pCMV, and pTK as positive control vectors. For high level constitutive expression of Luciferase, we recommend using the CMV (Cytomegalovirus) promoter vector and for low level constitutive expression of luciferase, we recommend using the pTK (thymidine kinase) promoter vector. 

See the table below for the various luciferase vector options we offer:

We offer three vectors with commonly used promoters built into the vectors:

• pEF1a-Tluc16-DD Vector (Human Elongation Factor-1 alpha; Cat. No. 88241)
• pNFkBTluc16 DD Vector (Nuclear Factor-kappa B; Cat. No. 88246)
• pCRE Tluc16 DD Vector (Carbapenem-resistant Enterobacteriaceae; Cat. No. 88247)

Vector-specific information can be found in the lot-specific Certificate of Analysis on the website

Thermo Scientific™ M-PER Mammalian Protein Extraction Reagent (Cat. No.78503) may be used to prepare lysate from washed adherent or pelleted cells, and then a portion of the lysate can be used to assay for beta-galactosidase activity.