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?
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.
- How the reaction works
- The purpose of the luciferase assay
- What you’ll need (instruments, supplies and how to choose)
- Deciding whether you need the luciferase assay kit
- The basic steps of the luciferase assay
- Which luciferase assay method to choose
- Additional resources
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.
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.
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. Luciferin Handbook. Luciferin Handbook. GoldBio provides most of what you’ll need including very high-quality luciferin for the best price around.
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.
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?
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 D-Luciferin In Vitro Protocol Handbook 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 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
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.
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).
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:
|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.
|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 ...
|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.
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