Thermo Scientific™ Pierce™ Firefly Luciferase Flash Assay Kit

Catalog No.	Quantity
PI16175	1000 Reaction Kit
PI16174	100 Reaction Kit

Catalog No. PI16175 Supplier Thermo Scientific™ Supplier No. 16175

Description

Assay regulatory element transcriptional activity in whole cell lysates with this highly sensitive flash-based firefly luciferase kit.

The Thermo Scientific™ Pierce Firefly Luciferase Flash Assay Kit provides reagents to measure Red firefly luciferase activity in whole cell producing a very strong flash signal which decays rapidly.

Thermo Scientific Pierce Firefly Luciferase Flash Assay Kit contains reagents to measure the activity of firefly luciferase in mammalian whole cell lysates. Containing a convenient lysis buffer, assay buffer and firefly substrate, this kit provides a complete reagent system for the intracellular detection of promoter or pathway activity. Light output resulting from the luciferase reaction is captured using a luminometer and can be correlated with the amount of firefly luciferase protein produced and used to determine the activity of the promoter driving firefly expression. These reagents are optimized to work with Thermo Scientific Firefly Luc Plasmids; however, the Flash Assay Kit may be used to detect the activity of other firefly or ATP-dependent luciferases utilizing D-luciferin as a substrate.

Highlights

• Highly sensitive detection of firefly luciferase activity
• Use with Pierce Firefly Luciferase Signal Enhancer for best results (maximum signal)
• Working solution stable for up to 2 months at -20°C
• Assay reagents compatible with other firefly luciferases
• Amenable to automation
• Contains a universal cell lysis buffer and optimized flash assay reagent
• Safe non-radioactive assay

Requires

Firefly luciferase and luminometer or other instrument capable of monitoring luminescence, such as the Varioskan ALF or the Varioskan LUX microplate readers; For best results (maximum signal), use with Thermo Scientific Pierce Firefly Luciferase Signal Enhancer

Recommended applications

Promoter studies for analyzing cis- regulatory elements and trans-acting factors; Drug screening; siRNA and miRNA screening; Multiplexed assays to study off-target effects; Secretory pathway/protein localization reporter assays; Signal transduction pathway analysis; RNA splicing studies

Specifications

• Type: Assay Kit
• Target: Luciferase, Firefly Luciferase
• Quantity: 1000 Reaction Kit
• Content And Storage: • Firefly Flash Assay Buffer, 5 mL, store at 4°C
  • D-Luciferin, 3 mg, store at 4°C
  • 2X Cell Lysis Buffer, 6 mL, store at room temperature
  
  Upon receipt store kit at 4°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: D-Luciferin

Frequently Asked Questions (FAQs)

I got a high background signal with a Firefly Luciferase Glow/Flash assay. Why is this?

This could be due to contamination of the control sample. Make sure to use new sample and change pipette tips after each well.

I got a high signal with a Firefly Luciferase Glow/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 
Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 µL per assay

What could be causing no signal or low signal with the Firefly Luciferase Glow/Luciferase Flash assay? Do you have any suggestions to get around this problem?

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.
- D-luciferin auto-oxidized: Protect substrate from light and maintain 100X D-luciferin at -20 degrees C; Prepare new Working Solution if used longer than 4 hours.
- Low luciferase expression: Use Pierce Firefly Signal Enhancer (100X) (Cat. No. 16180); Lyse cells in a 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 the volume of sample and reagent per well.
- Degraded luciferase protein: Store cell lysates on ice and perform assays immediately following cell lysis.

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. 

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.