Thermo Scientific™ TurboLuc™ Luciferase One-Step Glow Assay Kit

Catalog No.	Quantity
PI88263	100 Reactions

Catalog No. PI88263 Supplier Thermo Scientific™ Supplier No. 88263

Description

Measure TurboLuc™16 (Tluc16) luciferase activity in mammalian cells with a single reagent-addition step that is for high-throughput screening (HTS) applications using the Thermo Scientific™ TurboLuc™ Luciferase One-Step Glow Assay Kit.

The Thermo Scientific TurboLuc Luciferase One-Step Glow Assay Kit measures TurboLuc16 (Tluc16) luciferase activity in mammalian cells with a single reagent-addition step, making it ideal for high-throughput screening (HTS) applications.

• Sensitive - extremely bright, Tluc16 luciferase allows detection at a single copy per cell
• Increased dynamic range - optimized reagents result in higher signal-to-noise ratio
• Stable signal - less than 40% signal loss over 60 min duration
• Convenient - one-step, mix-and-read, scalable protocol
• Automation-friendly - amenable to HTS protocols
• Simple - does not require a luminometer with injectors

This glow-type luciferase assay kit is designed for use with cultured mammalian cells expressing the Tluc16 luciferase reporter enzyme. Tluc16 is a 16 kDa, novel intracellular luciferase derived from the marine copedod Metridia luciferase family. The wild type luciferase has been modified to reduce its size, increase its brightness, enable its intracellular expression, and optimize it for HTS. A series of vectors with and without promoters are available for expression of Tluc16 luciferase reporter in mammalian cells.
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 kit provides a highly sensitive bioluminescent reporter assay system for the detection of promoter or pathway activity.
Requires:
Tluc16 luciferase assay system and luminometer or other instrument capable of monitoring luminescence, such as Thermo Scientific Luminoskan™ Ascent and Varioskan™ Flash microplate readers.
Applications:

• Gene reporter assays
• Promoter studies for analyzing cis- and trans-acting regulatory elements
• Drug screening
• Multiplexed assays to study off-target effects
• Cell signaling or signal transduction pathway analysis

Specifications

• Type: Assay Kit
• Target: Luciferase, TurboLuc™18 Luciferase
• Sufficient For: 100 Reactions
• Quantity: 100 Reactions
• Content And Storage: TurboLuc One-Step Substrate (50X), 1 x 0.2 mL
  TurboLuc One-Step Assay Buffer, 11 mL
  
  Store in ultra-cold freezer (-68 to -85°C).
• Assay: Reporter Enzyme, Luciferase Reporter Assay
• Compatible Cells: Mammalian Cells
• Detection Method: Bioluminescence
• For Use With (Equipment): Luminometer (Microplate)
• Format: 384-well plate, 96-well plate
• Label Type: Enzyme Labeled
• Substrate Properties: Chemical Substrate
• Technique: Enhanced Chemiluminescence
• Substrate Type: Luciferase Substrate
• Substrate: Coelenterazine

Frequently Asked Questions (FAQs)

Do you offer an alternative product to TurboLuc Luciferase One-Step Glow Assay (Cat. No. 88263)?

Unfortunately, we do not have an alternative product for the discontinued product TurboLuc Luciferase One-Step Glow Assay (Cat. No. 88263).

With my TurboLuc Luciferase One-Step Glow Assay, I have been noticing that the signal is high and quickly decays. Why is this?

Here are possible causes and solutions:

- High luciferase expression: Reduce incubation time before collecting samples; Adjust instrument parameters to capture less signal (instrument-dependent); Decrease the amount of plasmid transfected into cells or decrease cell number.
- Control sample contaminated: Change pipette tips after each well.

What can cause low or no signal in the TurboLuc Luciferase One-Step Glow Assay?

Here are possible causes and solutions:

- Low transfection efficiency: Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein); Verify plasmid DNA quality using restriction digestion and agarose gel electrophoresis - use only transfection grade DNA; Note: Most high-quality plasmid DNA should be supercoiled; Use actively dividing, low passage cells; Use a different cell type.
- No promoter induction: Incubate cells under promoter-specific inducing conditions; Incubate the cells for a longer time after treatment; Change growth conditions to improve expression; Use a different promoter or cell type.
- TurboLuc One-Step Substrate auto-oxidized: Protect substrate from light and air and store at -80 degrees C; Prepare Working Solution immediately before use and protect from light.
- Wrong substrate used: Use only substrate supplied with the kit. Coelenterazine from related Luciferase kits (e.g., Renilla, Luciferase) will provide suboptimal performance.
- Low luciferase expression: Use a different promoter or growth conditions to improve expression; Adjust instrument parameters to capture more signal (instrument-dependent); Plate larger number of cells.

Does serum concentration have an inhibitory effect on the luciferase assay?

For Fetal Bovine Serum (FBS), there is no difference between 10% and 5% FBS. It is worthwhile to note that the type of serum does affect the luciferase assay. We tested the effect of 6 different types of sera (FBS, heat-inactivated FBS, dialyzed FBS, charcoal-treated FBS, donor adult bovine serum, and donor equine serum) on secreted luciferases, e.g. Gaussia, Gaussia-Dura, and Cypridina Luciferases. No noticeable difference was observed among different types of sera except for donor adult bovine serum. Unknown factors in donor adult bovine serum caused up to 35% inhibition in secreted luciferase activity. Therefore, we don’t recommend using donor adult bovine serum for Gaussia, Gaussia-Dura, and Cypridina Luciferases.

I used a white plate to read the luciferase assay and have very high background readings. Should I use a black plate?

The problems with white plates are related to cross-talk blocking, surface reflectivity and tendency to phosphorescence. We do not recommend using white plates because they give much higher background even if adapted to darkness for 10 minutes. The white plastic does not block the light completely, and some portion of the signal will go through the plastic and will be seen when you measure adjacent wells. The typical situation is if you change from black to white plates, where the background is about doubled and the signal is increased about 10 times. Black plates are recommend for best signal to noise ratio even though the RLU values will be lower. 

What is the benefit of using the TurboLuc Luciferase One-Step Glow Assay Kit over the Luciferase Glow Assay kits?

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.

What is the stability of the secreted luciferase?

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.

How quickly is luciferase secreted?

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.

What instrument filters are required to read the luciferase assays?

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.

Which luciferase assays can be used to specifically test media as a sample type?

Gaussia and Cypridina Luciferase assays can 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. 

Which luciferase assays do you offer to screen cell lysates and media?

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 lysates as 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.

Do you still offer quantitative IP (qIP) Luciferase assay kits?

Our qIP Luciferase assay kits have been discontinued but we do carry epitope-tagged (HA or c-Myc) and Tluc (TurboLuc luciferase)-tagged mammalian expression vectors, and qIP protein interaction assay reagents for these assay kits.