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Thermo Scientific™ Pierce™ Renilla-Firefly Luciferase Dual Assay Kit

Catalog No. PI16186
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PI16186 1000 Reaction Kit
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Catalog No. PI16186 Supplier Thermo Scientific™ Supplier No. 16186
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Description

Simultaneously detect intracellular Renilla and Red Firefly luciferase activity in mammalian whole cell lysates.

The Thermo Scientific™ Pierce Renilla-Firefly Luciferase Dual Assay Kit provides the necessary reagents to simultaneously detect intracellular Renilla and Red Firefly luciferase activity in mammalian whole cell lysates.

The Pierce Renilla-Firefly Luciferase Dual Assay Kit is a highly sensitive assay that allows for the simultaneous detection of Green Renilla and Red Firefly luciferase activity. Green Renilla luciferase acts as an experimental reporter with constitutively active red firefly 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.

Highlights

  • Simultaneous—concurrent, filter-based, wavelength-separated detection of two luciferase activities
  • Sensitive—measure Green Renilla and red firefly luciferase activities in the same sample
  • Fast—no quenching step required, unlike in traditional sequential dual assays
  • Multiplex—capable of quantitating two cellular activities in the same sample(s)

Requires

Green Renilla and Red Firefly luciferase reporters; filter sets at 525 nm±20 nm for Green Renilla Luc and 615 to 675 nm for Red Firefly Luc; luminometer or other instrument capable of monitoring luminescence, such as the Varioskan ALF or the Varioskan LUX microplate readers; injector for assessing more than 24 wells at a time

Recommended applications

Study two regulatory elements at the same time; monitor two signaling pathways simultaneously; enable studying more than one target per screen (e.g., off-target effects)

Specifications

Type Assay Kit
Target Luciferase, Firefly Luciferase, Green Renilla Luciferase
Sufficient For 1000 Dual Luciferase Assays
Quantity 1000 Reaction Kit
Content And Storage Sufficient For: 1000 dual-luciferase assays in microplate wells
• Renilla-Firefly Dual Assay Buffer, 50 mL (store at 4°C)
• 100X Coelenterazine, 0.5 mL (store at -80°C)
• D-Luciferin, 30 mg (store at 4°C)
• Luciferase Cell Lysis Buffer (2X), 60 mL (store at room temperature)

Upon receipt, store kit at -80°C, or store individual components as indicated above.
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
Product Line Pierce
Substrate Coelenterazine, D-Luciferin
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Frequently Asked Questions (FAQs)

What are the advantages of using TurboFect Transfection Reagent (Cat. No. R0533) over other transfection reagents with the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186)?

The Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186) luciferase vectors were optimized with TurboFect. However, it should be feasible to replace TurboFect with another transfection reagent if your cells tolerate the transfection reagent better and give you acceptable transfection rates.

Can I store my Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186) cell lysates at -20 degrees C to test later?

The stability of the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186) may be inconsistent from sample to sample at -20 degrees C. We recommend snap-freezing lysates at -80 degrees C with protease inhibitors to help prevent protein degradation.

Can I use the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186) with a luminometer that is not equipped with reagent injectors?

No. All Luciferase Dual Assay Kits, such as the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186), are flash-based assays. Flash luminescence reactions give a very bright signal over a short reaction time. Luciferase assays that are flash-based require the use of a luminometer or other luminescence-monitoring instrument equipped with automated reagent injectors.

I got a high background signal when I used a Pierce Gaussia/Cypridina/Renilla -Firefly Dual Assay Kit. Why is this?

Here are possible causes and solutions:

- Non-specific oxidation of substrate: Use a new sample; Avoid repeated freezing and thawing of the sample.
- Control sample is contaminated: Change pipette tips after each well; Reduce shaker speed during the cell lysis step to avoid contaminating the wells.
I got a high signal when I used a Pierce Gaussia/Cypridina/Renilla -Firefly Dual Assay Kit. Why is this?

This could be due to high luciferase expression. Here are some suggestions:

  • Reduce incubation time before collecting samples. 
  • Decrease the integration time on the instrument.
  • Dilute the sample: 
    Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 µL per assay.

    •
    I got a low signal in my lysate when I used a Pierce Gaussia/Cypridina/Renilla-Firefly Dual Assay Kit. Why is this?

    Here are possible causes and solutions:

    - Low luciferase expression: Lyse cells in smaller volume of 1X Cell Lysis Buffer; Use a different promoter or growth conditions to improve expression; Increase the integration time on the instrument; Scale up volume of sample and reagent per well.
    I did not get any signal when I used a Pierce Gaussia/Cypridina/Renilla-Firefly Dual Assay Kit. Why is this?

    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 - please note that most high-quality plasmid DNA should be supercoiled; Use actively dividing, low-passage cells; Use a different cell type.
    - No promoter induction: Incubate cells using promoter-specific inducing conditions; Incubate cells for a longer time after treatment; Change growth conditions to improve expression; Use a different promoter.
    - Substrate auto-oxidized: Protect substrate from light and air; Maintain 100X Coelenterazine at -80 degrees C, 100X Vargulin at -20 degrees C, and 100X D-Luciferin at -20 degrees C.
    I got a high background signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What went wrong?

    Here are possible causes and solutions:

    - Non-specific oxidation of substrate: Use less serum in the cell culture media; Note: Albumin can increase the auto-oxidation of Vargulin; Avoid repeated freezing and thawing of the sample.
    - Control sample is contaminated: Use new sample; Change pipette tips after each well.
    I am getting a high saturating signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What could have happened?

    This could be due to high luciferase expression. Here are some suggestions:

  • Reduce incubation time before collecting samples. 
  • Decrease the integration time on the instrument.
  • Dilute the sample: for secreted Gaussia/Cypridina Luciferase, dilute the sample using media from the cell culture; for cell lysate, dilute the sample using lysis buffer 


    • Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 µL per assay.
    Why am I getting a low signal in lysate using a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay? Can you provide some suggestions?

    Here are possible causes and solutions:

    - Non-optimized lysis buffer used: Assay luciferase activity in the media to confirm good expression of luciferase; Use only the provided lysis buffer.
    - Low luciferase expression: Lyse cells in smaller volume of 1X Cell Lysis Buffer; Use a different promoter or growth conditions to improve expression; Increase the integration time on the instrument; Scale up volume of sample and reagent per well.
    I got a low signal in media when I used a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What happened?

    Here are possible causes and solutions:

    - Insufficient luciferase accumulation in media: Incubate cells for a longer time.
    - Low luciferase expression: Use less media per well during the experiment;.Use a different promoter or growth conditions to improve expression;.Increase the integration time on the instrument;.Scale-up the volume of sample and reagent per well.
    - Treatment interfered with cellular secretory pathway: Transfect cells with a plasmid for constitutive expression of Luciferase (i.e., with pTK or pCMV promoter); Determine if luciferase actively expresses in media without treatment. Add treatment; Determine if there is a corresponding drop in luciferase activity from the constitutively expressed plasmid.
    I am getting no signal with my Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. Why is this?

    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 - use only transfection grade DNA; Use actively dividing, low passage cells; Use a different cell type.
    - No or low promoter activity: Use conditions known for promoter activation; Incubate cells for a longer time; Change growth conditions to improve expression; Use a different promoter.
    - Substrate auto-oxidized: Protect substrate from light and air; Maintain 100X Coelenterazine at -80 degrees C; maintain 100X Vargulin at -80 degrees C; Prepare new Coelenterazine Working Solution if used longer than 8 hours; Prepare new Vargulin Working Solution if used longer than 2 hours
    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. 
    Can the Promega CHECK-2 vector, PR-c8021 be used with our Pierce Renilla-Firefly Luciferase Dual Assay Kit (Cat. Nos. 16185, 16186)?

    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. 
    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.

    For Research Use Only. Not for use in diagnostic procedures.