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Coelenterazine

Posted by Karen on April 18th, 2017  ⟩  0 comments

All right, so you’re starting a new project that requires you to use the luciferase assay, and this is your first time. You might have a lot of questions. You might also have a lot of assumptions or misconceptions. So what do you need to know in order to get started? What should you look out for?

  A Crash Course on Luciferase Assays

Relax, sit back. We’ve got you covered. Scroll through our luciferin/luciferase crash course and get the answers to some of your most immediate questions about the luciferase assay.

What’s Covered:







What Is The Luciferin/Luciferase Reaction – How Does It Work?

In nature, bioluminescence occurs when chemical energy is converted into light. Many organisms such as fireflies, fungi and sea organisms use this process for a variety of reasons. The process occurs when luciferase catalyzes the oxidation of luciferin resulting in the emission of light.

The actual reaction below shows the process where luciferin is catalyzed by luciferase in the presence of ATP, oxygen and magnesium. The result yields oxyluciferin, CO, AMP and light.

Luciferin luciferase reaction - a crash course on the luciferase assay





What Is The Primary Purpose Of The Luciferase Assay?

Luciferase is a great way to test the strength and activity of a promoter. If, for example, you wanted to research the transcription within a promoter region, you can put the luciferase gene behind the promoter. When the gene gets transcribed, you will know whether or not you have a strong promoter based on the amount of light produced. More light produced in the assay means more luciferase was transcribed, which means you have a stronger promoter.

Another question you might have is when would you choose the luciferase assay and when would you choose qPCR if you’re examining gene expression. Here’s what to keep in mind: The qPCR method is going to measure your gene’s transcripts. It’s not going to tell you about the control of transcription. With luciferase, however, you can measure promoter activation and transcriptional regulation. Another thing to consider is how deep you want to look at your gene regulation. You might find that performing both techniques are a must in order to understand major aspects of your project.





What Am I Going To Need For The Luciferase Assay?

Before I talk about what you’ll need to perform the luciferase assay, I want to highlight something in case this is still very new territory. The luciferase assay is performed within the cells. Bioluminescence studies with the luciferin-luciferase reaction can also be performed in other models such as mice; however, in that example, it’s called bioluminescence Imaging. BLI still uses luciferin, but the instruments you use and protocols you follow will be significantly different. This article only covers information about the luciferase assay and does not go into more detail about BLI.



Reagents:

You’re going to need your reagents. You can either use a kit, which supplies you with everything you need or shop for the reagents a la carte.

For the necessary reagents if you plan to do this a la carte, refer to either our in vitro Luciferin Handbook. GoldBio provides most of what you’ll need including very high-quality luciferin for the best price around.



Equipment:

Luminometer

As far as equipment, you’ll need a luminometer. You may already have one, and if so, take note of whether it’s a microplate reading luminometer or a single tube luminometer. Find out if your instrument has injectors or not. And find out what wavelengths your instrument works on and at what temperatures. This is all going to help you further down in planning your experiment.

The microplate luminometers allow you to read samples in well plates. This can range usually between 96 and 384. The single tube luminometer, on the other hand, is going to read a single microcentrifuge tube.

Luminometers with injectors are important when you’re working with a flash type assay involving several samples. The flash luciferase assay kits are very common and provide higher sensitivity; however, they have a short half-life. In order to get a consistent read in time, the injectors inject the luciferin into your sample immediately.

If you don’t have injectors in your luminometer, that’s fine. I’ll address that later in the article.



Well Plates/ Tubes

Now you know what kind of luminometer you have, or the one you’re going to buy or borrow. You’ll also need tubes or well plates depending on what instrument you choose. 

luciferase assay crash course - luciferase assay for dummies - choosing your assay

When it comes to well plates, you’re going to encounter some choices here: flat bottom plates, clear plates, white or opaque plates, white plates with clear bottoms and so on.

Flat bottom plates are a must for this assay type (do not use round bottom well plates). They were especially designed for optical measurements and cell culture applications. More information about the flat bottom plate or F-bottom plate can be found at the well plate site. This website also has a more comprehensive list of the different well plate bottoms and what they are designed for.

Clear well plates allow you to see your lysates, but the drawback is that you can get background luminescence from neighboring wells. White well plates prevent that background; however, you can’t see you’re the lysates when you’re working with them. The white plates with clear bottoms are a solution to the visibility and background issues, but they can be expensive. Just keep those factors in mind in deciding what route to take.



What Luciferase Assay Kit Pack Size Should I Buy

The way to answer this question is to understand what constitutes an assay. For example, with our Luciferase Assay Kits, we have kits ranging from 50 assays to 10,000 assays. The question we have encountered is, “does that mean 10,000 plates or 10,000 tubes/wells in a plate?” One assay is one tube (one well/one reaction). Therefore, think about your experiment and the requirements you’re going to have.

Whether you’re using a single tube or a 96-well plate, the volumes used in our protocols will be the same. The protocol is written to accommodate a 96-well plate, but this can just as easily be used in a tube.





Do I Need To Use The Kits? What Do The Luciferase Assay Kits Include?

luciferase assay guide information - do you need to buy a kit?

This is a very simple answer: You don’t need the kits. You can order the luciferin, the ATP, and everything else, and then follow the protocols in order to perform the experiment. If you’re doing your work in vitro, then our D-Luciferin In Vitro Protocol Handbook will be very helpful.

If you are shopping for individual products, then we encourage you to consider quality, especially when it comes to your luciferin. The difference in purity can have an impact.

Other considerations when choosing luciferin can be found in this article, which goes in some detail about solubility considerations, assay considerations and more.

However, the kits present considerable convenience. For example, you don’t have to make the buffer since it’s already provided in the kit. When making your luciferin stock solution, there is no weigh out required because your luciferin is already measured. It also provides uniformity in your experimental setup.

Another advantage the kit offers is clarity on the products you need. For example, you may have almost all the individual products you need except for the luciferin and the buffer. If you’re new to the luciferase assay, you might be unsure about which luciferin to choose (sodium, potassium, free acid, etc.). You might also be unsure about which buffer to choose or how to make the buffer. The kit spares you from a lot of confusion and additional research on what to buy. However, should you need to purchase accessory products, our team is here to help you sort out what you need.

Note: GoldBio does not sell the reaction buffer individually. It is included in the kit, however. Our 5X Luciferase Lysis Buffer is a lysis buffer only. You can refer to pages 4-6 of the In Vitro D-Luciferin Handbook for instructions on how to make the reaction buffer.

Your approach to this decision is going to depend on what you have time for, what you might already have in the lab, what you feel like doing and don’t feel like doing, and what you can spend.

The kit components will vary based on which kit you choose. For example, the IlluminationTM Series Firefly & Renilla Luciferase Enhanced Assay Kit by GoldBio comes with: 5X passive lysis buffer, firefly luciferase assay buffer, GoldBio’s d-luciferin, renilla luciferase assay buffer and enhanced coelenterazine. 





What Are The Basic Steps Of The Luciferase Assay?

The steps of the luciferase assay are going to remain very similar whether you’re doing a dual reporter assay or a single reporter.

Step 1: Choose your luciferase reporter gene (firefly luciferase or renilla luciferase, etc.). I’ll get into the different methods which will factor into your choice further in this article. But for the time being, just remember that if you’re doing a dual reporter assay, your luciferases need to have different spectral measurements.

Step 2: Clone your reporter into your plasmid. If you’re doing a dual reporter assay, then you will clone your other reporter into a separate plasmid.

Step 3: Cotransfect your experimental cells with your plasmid.

Step 4: After an incubation period of 24-48 hours, remove your media and lyse your cells.

Step 5: Add the buffer containing luciferin to the lysate. The light from this reaction can be measured with the luminometer.

These are the general steps you can expect to follow. Your method is going to vary to some degree based on the type of assay you’re performing and the objectives of your experiment.





Which Luciferase Assay Method Do I Choose?

There are different types of luciferase assays to choose from. There are flash types and glowing assays. You could also perform a single assay or a dual luciferase assay (in rare cases, even a triple). You might be wondering how to choose. This is all going to depend on what you need for your experiment.



Flash Assays vs. Glow Assays

Flash

The flash type luciferase assay, which is the most common assay type, means that upon adding your substrate, the reaction is going to happen very fast. You have a very limited amount of time to add the substrate and measure the light emission. When working with a single assay (tube), this won’t be much of a problem.

Let’s look at a hypothetical scenario where you would be working with a 96-well plate doing a flash assay. Because the assay runs so fast, if you were using a multichannel pipette, but the time you finish pipetting substrate into the final wells, you’ll have lost maximum sensitivity in your first wells. It would be impossible in that setup to get an accurate reading. Another option, when running your experiment with a 96-well plate is to pipette substrate into a single well, get a reading, and then move on to your next well – 96 times. This is possible to do, but it’s going to take considerable concentration and patterning in your behavior to get the consistent results you need.

As mentioned earlier in this article under the equipment section, some luminometers that measure the light emission from this reaction have been designed with injectors that automate the process of adding substrate, making it immediate and consistent. This ultimately solves the problem you face when working with a lot of samples in a short period of time. But luminometers with injectors require more substrate for an experiment. The reason is because some substrate is always lost, and it prevents the potential for running out.

The benefit of a flash type luciferase assay is that it produces very sensitive results. If this is extremely important for your experiment, and you have the equipment to carry it out, this is the type of luciferase assay you want to choose.



Glow

Maybe you don’t have a luminometer with injectors, but you’ve got a lot of samples to work with at a time and you want to ensure consistent results. The glowing luciferase assay is an alternative that buys you time. GoldBio’s Dura-Luc Lyophilized Firefly HTS Assay Kit has a half-life of nearly 3 hours. This allows you to pipette substrate into several wells before the signal fades. Another huge benefit is that it lets you compare results over multiple plates. The glow assay provides you with the accurate, consistent read your experiment needs. The downside, though, is that it is not as sensitive as the flash type.



Single Reporter Assays vs. Dual Reporter Assays

Single Reporter Assay

The reason you might lean toward a single reporter assay is because it cuts cost and time when studying expression. In the single reporter system, you would be using only luciferin (or coelenterazine if you’re working with renilla) as your substrate, and measure emission from that alone.

The drawback to only doing this is the lack of normalization. It’s not going to produce as detailed results as using the dual reporter assay.



Dual Reporter Assay

The dual assay system is most commonly performed with firefly and renilla luciferase. The dual system improves your overall accuracy by normalizing your data.

In this system, one reporter (e.g. firefly luciferase) will look at the experimental promoter activity. The other (e.g. renilla luciferase) is going to be used as your control for transfection efficiency. Therefore, in this experiment, your green firefly luciferase is going to measure experimental conditions, while your blue renilla luciferase is going to be connected with a constitutive promoter, measuring transfection and cell viability. The order can be reversed and firefly luciferase can be used as your control instead.

When performing the dual reporter assay, it’s important to choose reporters with spectral differences (different wavelength emission) in order to get an accurate read.



What to choose

Ultimately, this depends on what you need for your experiment. If you need the setup to be highly accurate and detailed, use the dual-system.

Outside of common practice, researchers have used the dual reporter system to shed light in other, innovative ways (some researchers have even used a triple reporter system).



Additional Resources

This article will only give you a little more clarity on the project ahead. But fear not. GoldBio’s handbooks and other articles might help address questions that arise. Below is a list of other helpful resources that might become useful later down the road:

Resources Description
Luciferin In Vitro Handbook Details the preparation and steps for working with luciferin in in vitro settings.
Luciferin In Vivo Handbook Details the preparation and steps for working with luciferin in in vivo settings.
Beetle vs Firefly Luciferin Firefly luciferin is pretty common, but you might be also hearing “beetle luciferin.” What’s the difference? This article sorts that out.
Luciferin FAQ This FAQ page lists the most common questions pertaining to luciferin.
10 Things and Beyond to Consider When Shopping or Using Luciferin/Luciferase
If you find yourself questioning the difference between various luciferase or luciferin types, this guide will set it all straight. Find out what to look out for when shopping for ...

Does My Chemical's Purity Really Matter?

One of the questions we receive at GoldBio is whether purity really matters when it comes to chemicals. In this article, we go into detail about why it does matter, even examining what a small percent difference can do to luciferin.


References:

96-Well Plate Bottom Shapes - Difference Between Bottom Shapes. (n.d.). Retrieved March 22, 2017, from http://www.wellplate.com/96-well-plate-bottom-shap...

Carceles-Cordon, M., Rodriguez-Fernandez, I., Rodriguez-Bravo, V., Cordon-Cardo, C. and Domingo-Domenech, J. (2016). In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells. Bio-protocol 6(6): e1762. DOI: 10.21769/BioProtoc.1762; Full Text

Differences between in vitro, in vivo, and in silico studies. (2012, January 03). Retrieved March 24, 2017, from https://mpkb.org/home/patients/assessing_literature/in_vitro_studies

F-Bottom Shape - Flat Well Bottom - Precise Optical Measurements. (n.d.). Retrieved March 22, 2017, from http://www.wellplate.com/f-bottom-shape/

Khan, F. (2013, August 26). The Luciferase Reporter Assay: How it works. Retrieved March 23, 2017, from http://bitesizebio.com/10774/the-luciferase-reporter-assay-how-it-works/

Ling A, Soares F, Croitoru DO, et al. Post-transcriptional Inhibition of Luciferase Reporter Assays by the Nod-like Receptor Proteins NLRX1 and NLRC3. The Journal of Biological Chemistry. 2012;287(34):28705-28716. doi:10.1074/jbc.M111.333146.

Smalle, T. (2010, May). Luciferase Assay. Retrieved March 24, 2017, from http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5421.long

U-Bottom Shape - Round Shaped Well Bottom - 96-Well Microplate. (n.d.). Retrieved March 22, 2017, from http://www.wellplate.com/u-bottom-shape/


    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science. 



Category Code: 79104 88231 79107 79109 88251

Posted by Chris on June 27th, 2013  ⟩  0 comments

“Before, beside us, and above
        The firefly lights his lamp of love.”
                        by Bishop Reginald Heber

Bioluminescence is one of the premier tools that scientists have in research, whether studying in vitro or in vivo. Few devices allow for the range, versatility, and ease of use as our adaption of the firefly’s twinkling star. But the firefly luminescence was only the beginning, and biologists have found many other species (mostly in shallow, coastal waters) which have developed the ability to light their own way and which we can copy for our own use and benefit.

Firefly luciferase, with its substrate luciferin, is still by far the most popular system for use in bioluminescent imaging (BLI), with Gaussia luciferase, and its substrate coelenterazine, a close second. Over the years, these two systems have been combined in various methods or kits in order to provide a more expansive research device. Most often, one or the other is used as a system control while the other pulls the heavy load. And there have been many attempts to expand the system even further, such as altering the luciferase cDNA and changing its emission spectrum in order to add a third BLI wavelength. But most of this work has been done in vitro, where the “trouble” of dealing with more than one substrate in a system can be a burden. But in vivo researchers are less bothered by such minor complications.

In animal studies, there are other things to worry about. For instance, how well does a new system handle the body temperature of the model? Can the substrate get to the test site, how quickly/slowly, and which route of injection works best? Will the substrate be broken down in the system? Can we visualize the BLI through the thousand-fold layers of cells in the animal model? There have also been many combinations of BLI and fluorescence systems in order to expand the in vivo systems as well, but with many models displaying autofluorescence, the advantages of doing so is somewhat muted by comparison. Into that line of discovery, enter Dr. Casey Maguire and his group from Harvard Medical School/Massachusetts General Hospital.

Maguire et al. wanted to develop a system in which three different luciferase signals could help report cancer cells and their cellular interactions. Firefly and Gaussia luciferases were a given, but they needed another, and decided on Vargula (or Cyprindina) luciferase. This relatively new luciferase was found in Vargula hilgendorfii (previously called Cypridina hilgendorfii), a crustacean sometimes called a sea shrimp or sea-firefly. V-Luc (or sometimes C-Luc) utilizes a substrate called Vargulin to produce a blue colored light around the 450nm wavelength. Using a mouse model, Maguire injected cancer cells intracranially which had been modified with either VLuc, FLuc or GLuc cDNA. Ultimately, they wanted to test their ability to “monitor the effect of an adeno-associated virus (AAV)-mediated soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) therapy against intracranial glioma tumors.”

The results were outstanding, barring a few caveats which you can read for yourself in the discussion section of their article. There was little to no overlap in the BLI signals between the three substrates and all three were clearly visible, even in deep tissue, like the brain. The use of luciferin, coelenterazine, and now vargulin, as triple BLI reporters makes for the best of a cost-effective, sensitive and easy-to-use reporting system. And at GoldBio, that’s just the way we like it!

Triple Bioluminescence

 
 

Maguire, Casey A., et al. "Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes." Molecular Therapy—Nucleic Acids 2.6 (2013): e99.

Category Code: 88241 88231

Posted by Chris on June 20th, 2013  ⟩  0 comments

If you’re anything like me (geek that I am), every new technological device tends to get your blood pumping and invokes an involuntary reflex to reach for your wallet. That’s even truer for the ever-popular “i”-products which tend to grab our collective-geek attention even faster with every new device. Now there are some clever scientists from the University of Bonn in Germany who have developed a new reporter system utilizing Gaussia luciferase, the “iGLuc”!

While researching the inflammasome process, and specifically IL-1β, a primary target of caspase-1, Bartok et al. hit a frustrating road block. Inflammasomes are large, multiprotein oligomers that are intregal parts of the immune response system. They are a platform which supports an inflammatory cascade after sensing damage-associated molecular patterns. Caspase-1 is an enzyme that’s utilized by the inflammasome cascade in order to proteolytically cleave specific proteins (such as IL-1β precursor) into active, mature peptides. Once cleaved, IL-1β can finally bind to its receptor in order to induce a variety of cellular responses, such as pyroptosis; a form of programmed cell death that is in response to inflammation.

Bartok was looking for a better way to analyze IL-1β. ELISA techniques were not sensitive enough to distinguish between the IL-1β precursors and mature IL-1β, and Western blotting was too time consuming and useless for high throughput analysis. So, instead they devised a fusion protein of pro-IL-1β and GLuc (Gaussia Luciferase) and called it iGLuc! Unexpectedly, they first saw virtually no luciferase signal from the fusion, even though they were seeing high expression levels of luciferase in the system. But they discovered that pro-IL-1β tends to form a protein aggregate which acts to restrict the release of the signaling C-terminal portion of GLuc. But with the simple addition of caspase-1, pro-IL-1β was cleaved and a corresponding bioluminescent signal could be measured.

The resulting process seems to make for an excellent reporter assay for inflammasome activity! Bartok tested the system both in vitro and in vivo and the system showed good sensitivity and specificity as well as a great signal to noise ratio. The system also shows a lot of promise that it can be further applied to other proteases as well! So, if you’re in the field of inflammasomes (or if you have to own every new device), be sure to keep an “i” out for the iGLuc system. It may become the next, best geeky thing on the technological front! You can find their complete article here.

iGLuc in vivo images

Category Code: 88221 88241

Posted by Patrick on November 12th, 2012  ⟩  0 comments

Coelenterazine is a naturally occurring substrate, or luciferin, for a diverse collection of luciferases.  In the presence of molecular oxygen, the luciferase oxidizes coelenterazine, generating a high-energy intermediate and emitting blue light in the process (1).  Unlike beetle luciferin-luciferase systems, coelenterazine luciferases do not require ATP and are often more convenient for in vivo bioluminescence experiments (2).  The best-characterized coelenterazine luciferase comes from the sea pansy Renilla.

Coelenterazine is also the chromatophore cofactor of another family of marine photoproteins, exemplified by aequorin from the Aequorea jellyfish.  In a variation of the luciferin-luciferase reaction, aequorin is “preloaded” with a stably bound, reactive form of coelenterazine and the oxidation reaction proceeds upon binding of Ca2+ to the aequorin protein.  This response to calcium is essentially oxygen-independent and the amount of blue light emitted by aequorin is proportional to the enzyme and Ca2+ concentrations (1).

Coelenterazine is hydrophobic and can easily cross cell membranes, making it amenable to use in whole-cell experiments.  In addition to native coelenterazine, several analogs of coelenterazine with altered emission properties or chemical stabilities are commercially available.  As a substrate for Renilla luciferase (Rluc), native coelenterazine emits light at ~460nm.  In comparison, coelenterazine 400a, a commonly used analog often referred to as DeepBlueC, emits light at ~395nm but decays rapidly in aqueous solution(3)(4).

The coelenterazine-aequorin  bioluminescence reaction is sensitive to even slight changes in Ca2+ concentrations and has been used extensively as a biosensor for intracellular calcium(5).  Unlike calcium-sensitive dyes, recombinant aequorin can be targeted to specific cellular compartments, allowing for subcellular resolution of calcium measurements.  Wild-type aequorin can be used to measure calcium concentrations that fall between 0.5µM-10µM and mutations that lower the protein’s affinity for calcium can expand this range up to 100µM(5).

Bioluminescence resonance energy transfer (BRET) is a recently developed experimental technique that also exploits coelenterazine-based  bioluminescence (6) (7). Like fluorescence resonance energy transfer (FRET), BRET involves the transfer of energy from a light-emitting donor to a fluorescent acceptor, resulting in a shift in the detectable light spectrum(8).  This energy transfer is only possible over distances less than 10nm, making it an effective gauge of protein interactions when the donor and acceptor proteins are fused to suitable targets.  Unlike FRET, BRET uses an enzymatic source of luminescence not external optical excitation, so it avoids several common FRET complications like photobleaching, autofluorescence, and tissue damage from laser exposure(8). BRET has been used successfully in bacteria, yeast, plants, and mammalian cells and has proven especially valuable for studying real time interactions, the mechanisms of G- protein coupled receptors, and high variation of the originally published BRET technique, uses Rluc and GFP, as the donor and acceptor respectively, and coelenterazine 400a as the substrate.

The BRET technique can be further optimized to suit a range of experimental systems, instrumentation, and conditions (6). The use of different coelenterazine substrates influences the quantum yield of the donor reaction, the overlap of donor and acceptor spectra, or the rate of signal decay.  Also, mutants of Rluc exist that alter its emission spectrum or increase its stability in cell culture conditions (2).

1.  Wilson T, Hastings JW. BIOLUMINESCENCE. Annu. Rev. Cell. Dev. Biol. 1998;14(1):197-230.

2.  Loening AM, Fenn TD, Wu AM, Gambhir SS. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Engineering, Design and Selection 2006 Sep;19(9):391-400.

3.  Pfleger KDG, Seeber RM, Eidne KA. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 2006;1(1):337-345.

4.  DeepBlueC is a trademark of the Packard BioScience Company.

5.  Brini M. Calcium-sensitive photoproteins. Methods 2008 Nov;46(3):160-166.

6.  Bacart J, Corbel C, Jockers R, Bach S, Couturier C. The BRET technology and its application to screening assays. Biotechnology Journal 2008;3(3):311-324.

7.  Pfleger KDG, Eidne KA. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Meth 2006 Mar;3(3):165-174.

8.  Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences of the United States of America 1999 Jan;96(1):151-156.

9.  Kocan M, Pfleger KD. Detection of GPCR/β-Arrestin Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer Technology [Internet]. In: G Protein-Coupled Receptors in Drug Discovery. 2009 p. 305-317.[cited 2010 Feb 2 ] Available from: http://dx.doi.org.laneproxy.stanford.edu/10.1007/9...

Category Code: 88221 88251