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Luciferin

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 Karen on April 23rd, 2015  ⟩  0 comments

It’s defrost day in the lab, and you think to yourself, “This might be the best day to organize my fridge.” Then you open the door, your jaw drops and you change your mind, choosing to simply deal with the defrost project rather than the organization. However, when it comes to your upright fridges and freezers that store delicate samples, enzymes and other reagents, organization is crucial to its efficiency. Loitering with the door open, attempting an impossible search for a product is not only a waste of energy and time, it’s also bad for the products stored within.


But guess what? You’re not alone with this problem. The household fridge is the original box of disorder. And it might not have ever occurred to you that the organizational tips of domestics might also be considerably useful in a scientific lab.

So here is your ultimate guide to keeping your laboratory fridges and freezers organized. It combines known lab suggestions with household suggestions. But it’s not foolproof. Maintenance and consistency is still going to be a personal challenge. And check out  our follow up blog which expands more on laboratory organization.


1.  Inventory Management:

  • Software Based: This is definitely your first step to organization, and it extends beyond the fridge. Thankfully, we live in the digital age and there is plenty of software available. Of course, if you’re on a budget we recommend trying Quartzy* for this. The reviews attest to its ability and it is provided at no cost to the user.
  • Spreadsheet/Cloud Based: If you’re a little partial to using spreadsheets, it might be useful to manage the spreadsheet on a Google Sheet because of its cloud based platform. That means anyone within your lab can access it at any time and log information – if you so choose that is. Of course, your institution may require an Excel submission of inventory for safety reasons. Thankfully, text in Google Docs can be easily copied and pasted. I still recommend keeping your inventory with Google due to its accessibility and ease of collaboration.
  • App Based: Primarily, the moral of this blog is that what can be used for home can sometimes be used for the lab. If you want to keep it simple or strictly for the fridge, browse your mobile device’s app store. There are several fridge/freezer management apps. While they’re dedicated to the household appliance, some can work equally well in the lab. For instance, the Freshbox app allows you to snap pictures of the product, set the shelf life, expiration date, add notes and set reminders for expiration. It’s simple, personal, easy to use, and has the perfect criteria needed for the management of your reagents. There are some drawbacks to the app, so find what works for you. 


 


2. Labels Labels Labels Labels:

Most of you are already doing this. The question is to what degree and how disciplined are you at updating and obeying the labels? If you share a fridge, it’s important to establish a categorical place for everything, and to have it labeled clearly. Underlings in your lab might be better about putting things back where they found them as long as the place plainly exists. Make sure you set up a place that can house multiple working tube racks or freezer boxes for those working under you in the lab. Have it labeled – perhaps even by name. On that note, if you’re not sure how to label the grated shelves of a fridge or freezer, consider using a suitcase tag. Even after the shelves frost over, the tag is hooked in place. And if you place labeled lab tape over the vinyl sleeve, your writing will remain visible.


3.  Storage Safety

While I trust you all are masters of lab safety, it is always worth the reminder. Make sure you store dangerous goods appropriately, being careful not to place products that might react with each other near each other. 



4.  Keep Like with Like: 

This is one of the golden rules of domestic organization. In some labs it might already be applied. In others, a fridge or freezer may have gotten so out of hand that anything is placed anywhere. Keep your enzymes with your enzymes, your samples near samples – however you want to categorize it, always keep like with like.


5.  One Shelf at a Time

The big tip from domestic experts is to take organization one shelf at a time. This is just as valuable in the home as it is in the lab. You might only have a small designated space in a reserve fridge to hold temperature sensitive products while you organize the freezer. So going one shelf at a time lets you make the best use of what little room you have. The other benefit is that you are not overly committed to the process. It’s a grueling task, and it’s easy to get in over your head. By taking it one shelf at a time, starting from the top and going down, you can easily free yourself from the job when necessary. If you’re doing this on defrost day, then I suggest you organize before you defrost.



     


6. Cleaning As You Go:

As you work from one shelf to the next, inventorying and trashing useless items, keep a cleaning cloth handy to wipe each shelf down. Don’t forget the walls and racks. 


7.  Square vs. Circular Containers

An interesting piece of domestic advice that is useful to your lab is storing items in square containers vs. circular containers. This is because circular containers do not make good use of corners and therefore waste more space than necessary. That isn’t to say you should avoid round containers, but consider squares as much as possible when reorganizing your fridge. For existing circular containers, find ways to stack them on top of a pre-existing stack of square containers. *For storing petri dishes, consider the tips provided in number 8.



8.  Everything Visible – Everything Accessible

Here’s the next golden rule for household fridge organization. And it can be extremely useful to apply in the lab. Not only do you hate pulling everything out of the freezer in order to find an old freezer box in the back, but you risk the warming and cooling of reagents or samples that might face some degradation. Instead, consider using Fridge BinzTM. These are great containers that are not only clear, but also allow you to pull out what is stored. With this tool, the back of the fridge search is no longer scary. I get it though, not everything is small enough to fit in these types of bins, so think inside a bigger box. Get some clear Rubbermaid tubs instead. They’ll give you the same benefit of visibility and accessibility.  Coolers and freezer boxes could be better stored in clear bins as well. As for sleeves of petri dishes, it wouldn't hurt to keep them in a larger, clear container such as these examples so that they can be neatly taken out and put back as needed.


9.  Turn Tables

Going back to accessibility, the revolutionary concept of the Lazy Susan doesn’t have to be limited to cabinets and tables at home. They’re useful in the fridges and freezers within households and laboratories. When Fridge Binz TM won’t work, a turn table might be your next best solution. You can find them anywhere and in a variety of sizes.


10.  Knowing the Anatomy of Your Fridge

Within a laboratory, you do have the advantage of having appliances designed specifically for your needs. High-end scientific fridges and freezers might be better suited for even distribution of temperature; however, not all of them are like this. If you aren’t aware of temperature zones in the fridge, our image below is a great illustration, detailing fridge temperature distribution. 

Here’s a big thing to consider, if your reagents or stock is sensitive to freeze-thaws, it may not be the best idea to store them in the arm! The arm is considered the warmest part of the fridge, and since the door is constantly opened with people often lingering, your samples, enzymes and stock solutions of ampicillin, for example, might be at more risk than you realized. Ultimately, you want to keep your items visible, accessible and protected.


You might now be thinking, “Well those tips are great, but I have 20 years of research in my fridge. Tell me how to fix that.” I have highlighted some additional tips to help with that in our follow up blog. Until then, recall the tips from number 7 and 8: Keep square containers and consider visible storage bins for old freezer boxes. It won’t be to the level of organization you desire, but it might be presentable and easily maneuvered around.


*It should be noted that GoldBio has its products listed on Quartyz; however, this mention was unsolicited.


    
              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: 79105 79109

Posted by Chris on March 6th, 2014  ⟩  0 comments

There is, I think, one thing that unifies nearly every species on Earth, a single constant that goes beyond similarities in DNA, genomes, cells or evolution. A “universal” tie-in which, even if you could genetically engineer and build from scratch an entirely new creation unbeholdened to any previous creature, rules every single body, great and small. Simply, we are all children of and are ruled by a planet with a 24 hour axis rotation.

This continuous cycle of light and dark creates the circadian rhythm that drives at the very core of our existence, mandating our waking and sleeping, whether human, insect, tree or bacteria. We live and die on the phases of our great home-world, and it takes thousands of years of isolation and/or specific adaption to overcome that basic, simple, biological drive. As humans, we often think that we have subjugated the world around us, but it’s abundantly clear to anyone who’s ever traveled long distances by plane (or even worked in a split-shift job) and suffered a “jetlag” effect, that we are still, quite obviously, slaves to our primal, internal clocks.

Despite our seeming dependence on it, we are still very much “in the dark” about how our circadian clock works. We know, for instance, that the clock is a function of the Suprachiasmatic nucleus (SCN), a small, rice-sized node in the hypothalamus that uses highly specialized retinal ganglion cells to detect light patterns, signaling genes such as Clock (Clk) and Period (Per) in the SCN to begin the oscillation of our internal clocks and maintain the speed of the oscillations over the next 24 hours. We know that there is an auto-regulatory feedback loop that prevents obtuse or episodic entrainment from occurring. But the way in which that regulation works hasn’t been as well understood...until just recently when a group out of Oxford led by Russell Foster and Stuart Peirson took an in depth look at the genes involved in the SCN feedback mechanism.

There is a real impetus to understand the way these genes process the external cues and self-regulate to govern our lives, and not just for jet-setting, world tourists, but also for the many people who suffer from circadian rhythm deficiencies. Foster and Peirson’s worked at the Per1 and Sik1 genes and how they affect CREB-mediated clock gene expression and that CRTC1 acts as a coactivator of the CREB transcription of those genes. They were able to show that knockdown of the Sik1 gene, both in vitro (with the smart use of Gold Bio Luciferin) and in vivo, enhanced the behavioral phase shifts and retrainment to new light-dark cycles by allowing the CRTC-continued transcription of Per1!

SCN Cirdadian Pathway

The role of the SIK1 negative-feedback mechanism is important to prevent our bodies from constantly readjusting to every minor change of stimuli that we’re faced with every day. Imagine if that streetlamp outside your window was suddenly able to change your body’s circadian clock to an opposite light-dark cycle or if, in the process of getting up in the middle of the night for a drink, the refrigerator light suddenly informed your body that it was morning and that you should stay awake? There is a definite need for the primary clock that we live by to keep itself in check, despite thousands of stimuli every day. That’s what the SIK1-CRTC pathway does…the proverbial time-keeping engineer on the train of life, keeping the system on time.

However there are always times when the clock goes haywire and needs a reboot. Currently, we don’t have one. But research into these mechanics and results like that of Foster and Peirson’s will someday make that a reality. And I can just about imagine the end to jetlag as we know it…but until then, I’ll just have to take a nap.





Jagannath, A., Butler, R., Godinho, S. I., Couch, Y., Brown, L. A., Vasudevan, S. R., Flanagan,K., Anthony, D., Churchill, G., Wood, M., Steiner, G, Ebeling, M., Hossbach, M., Wettstein, J., Duffield, G., Gatti, S., Hankins, M., Foster, R. & Peirson, S. (2013). The CRTC1-SIK1 Pathway Regulates Entrainment of the Circadian Clock. Cell, 154(5), 1100-1111.

Category Code: 79105 79102

Posted by Chris on August 1st, 2013  ⟩  0 comments

One of the questions we receive from time to time at GoldBio is whether purity really matters when it comes to chemicals. We can understand that question. There is a huge variety of “grades” with different purities available for almost every chemical on the market. Additionally, the grades/purities from one supplier do not always match exactly with that of a second supplier, so we know that it can be very difficult to compare otherwise seemingly equivalent products. What really are the differences between common grades such as Ultra Pure, ACS, Biotechnology, Reagent or HPLC? And which one should you use?

In a small lab, the answer is typically, “use whatever is already available!” But if you are designing an experiment from scratch or want the experiment done right, you want to make sure that the reagents you’re using are the right ones for the job. There are differences between the grade types that might be important for specific applications. For instance, "Ultra Pure" is a grade that signifies that the chemical has a purity exceeding more common grades. "ACS" is a grade for chemicals that specifically conform to the requirements of the American Chemical Society (for that chemical). However, Biotechnology Grade or Tissue Culture Grade might have an equal level of chemical purity (98-99% in some cases) as the Ultra Pure or ACS, but they are further purified to remove more specific contaminants, such as nucleases or bacteria, which could cause havoc in specific experiments. In those situations, are you really sure you want to use that ACS grade chemical (that’s been on the shelf for 3+ years and opened several dozen times) for your tissue culture experiment that might take you the better part of 3 months to conclude? Trust me, it really is better if you get the appropriately graded chemical.

Of course, chemical purity is still important. For instance, Goldbio’s Luciferin is 99.7-99.8% pure, the highest purity luciferin available on the market!** Most other suppliers have a minimum purity of only 98% (and sometimes, as low as 95%)! Maybe that doesn’t sound like much, but let’s do the math anyway. If 1 gram of 98.0% luciferin is dissolved in 25 ml buffer (a fairly standard dilution for in vivo assays) there would be 0.02 g of any potential contaminants. Again, that doesn’t sound like much, but that actually equates to 0.8 g per liter. If our theoretical contaminant has a molecular weight of 1000 g/mole, then the contaminant would be at a concentration of 800µM! That’s more than high enough to inhibit some enzymes in a cell. 

Luciferin purity is principally quantified by HPLC, and any impurities which may be left in the chemical are typically labeled as “unknown impurities”. The sensitivity and reliability of HPLC make common impurities fairly easy to distinguish and separate, so any unknown impurities may give you reason to pause. Of course, these unknown impurities may or may not be detrimental to an experiment. But just like in the previous example, are you really sure you want to trust a reagent with a lot of unknown impurities? Can you really be sure that potential impurities in your luciferin will be benign and have no side-effects on your 6-12 month mouse or rat study?

At GoldBio, we understand that question. It is why we strive to provide reagents, like luciferin, that are the best quality for your experiments. It is why we test our reagents personally to guarantee their efficacy. And that is why you trust GoldBio to provide those reagents for your research. 

It's just good science.

We would love to hear from you, so if you have any questions about any of our products, you can email us at: techsupport@goldbio.com!
 

  

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Posted by Chris on July 25th, 2013  ⟩  0 comments

Tuberculosis is a common, and often lethal, infectious disease which has circulated since the dawn of mankind. It is caused by the bacteria, Mycobacterium tuberculosis; a small, aerobic, nonmotile bacillus, which tends to lodge in the pulmonary system and from where it then spreads through coughing or sneezing respiratory fluids through the air. It has been found in the bones of Egyptian mummies and was once the cause of nearly 25% of all deaths in Europe. TB remains the world’s second most lethal, infectious disease (behind HIV) and has a significantly high rate of occurrence in African, South American and Asian countries, where the rate of death is anywhere from 250-3000+ per 100,000. The World Health Organization (WHO) estimates that roughly 1/3 of the world’s population have been infected with TB (although 90-95% remain asymptomatic) and about 1.5 million people die from it every year. The resurgence of the disease, and its drug-resistant varieties, led the WHO to declare it a global health emergency in 1993. Tuberculosis is also one of the major research areas to which the Bill and Melinda Gates Foundation fund every year, which awards nearly $800M (total) to researchers through its Global Health Program.

In the lab, Tuberculosis can also be difficult to screen, with a 16-20 hour replication (compared to under an hour for most other bacteria), and can take 3-4 weeks to form on solid media before any in vivo testing can be done. This can be a significant impediment for fast, de novo antibiotic research and Nuria Andreu, from the Department of Medicine at Imperial College London, and associates wanted to use luciferase to change the score!

That’s not to say that bioluminescence hasn’t been used on TB in the past. Researchers have been using BLI on TB strains for almost 20 years, but not necessarily with the live strain of M. tuberculosis, or with in vivo imaging in live mice. Andreu wanted to create a reporter gene with FFluc in a virulent M. tuberculosis strain. First, the FFluc would need to be modified to red-shift the signal so that the signal would be more thermostable. This was was accomplished by mutating 6 amino acids in the sequence to develop a signal that shifted the emission signal from 560nm to 620nm. Second, they had to develop an integrase-free reporter into M. tuberculosis to stabilize FFluc reporter signal over successive generations. The integrase-free reporter responded with greater than 99% retention of the reporter gene after 3 months of in vitro growth, compared with just 60% retention rate for the parent strain!

Ultimately, this study shows great progress in creating a virulent reporter strain of M. tuberculosis which can be useful in drug research. Andreu was able to detect the presence of the bacteria in the lungs of live mice after only 2 weeks post infection (105 cfu), and as few as 103 cfu were detectable ex vivo in both lungs and spleens of the mice after dissection. Most importantly, they were able to make a rapid assessment of antibiotic efficacy, treating the diseased mice with Isoniazid (an organic compound often used as the first line medication in treatment of tuberculosis). They were able to see a nearly 9-fold decrease in the BLI signal for treated mice versus the control group after only 7 days of treatment.

TB-Luciferase Drug Treatment isoniazid

Ultimately, this looks to be a great system for early lab development of new antibiotics. As resistance in TB continues to spread, we need new forms of treatment in order to stem the tide of resurgence and begin to eliminate the disease in the poorer sections of the world where TB remains rampant and the death toll remains high. Hopefully, this new reporter will help in developing those new drugs and we can begin to put an old disease to rest.

 
 

Andreu, N., Zelmer, A., Sampson, S. L., Ikeh, M., Bancroft, G. J., Schaible, U. E., Wiles, S., & Robertson, B. D. (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy.

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