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Posted by Fernanda on October 17th, 2018  ⟩  0 comments

Every day scientists in laboratories across the world sit at their desks and painstakingly design experiments in the hope of making a discovery that will change how we think about a biological process. Because biological processes such as enzymatic activity are dependent on pH, one critical aspect of the experimental design is choosing a buffering system that will help maintain a stable pH without altering the results.

And often, it is the choice of buffer that makes or breaks the experiment. It is possible that the buffer you are using in your lab might be the reason your experiment is failing. Here, you will find how a buffering system works, a description of the characteristics of a good buffer and a list of possible applications and characteristics of the most commonly used biological buffers.

Scroll to the bottom for printable guides

What is a buffer?

A buffer consists of a weak acid (proton donor, HA) and its conjugate base (proton acceptor, A -). In water, HA can dissociate into A- and H+. H+ then reacts with water to form H3O+. In the aqueous buffer solution, H3O+, HA and H+ exist in equilibrium with each other. The buffering mechanism consists of two reversible reactions where the concentration of proton donor and proton acceptor are equal.

  

Then, when a strong acid or base is introduced into this system by the scientist or by enzymatic activity during the experiment, the new ions from the introduced acid or base (H + or OH-) are absorbed by the buffer and the pH remains stable preventing changes in protein structure and function.

 

Buffers cannot arbitrarily moderate any changes in ion concentration. Their optimal buffering capacity, or range, is defined by the dissociation constant, or ka, of the acid. We commonly discuss buffering capacity in terms of the pKa or the logarithmic constant of ka. We consider the buffering capacity of a specific buffer to be the pKa ± 1. For example, a buffer with a pH of 6.8 has a pH buffering range of 5.8-7.8.

What is a Good biological buffer?

Years ago, scientists performed biochemical experiments with inadequate buffers that greatly limited the impact of their research. These buffers exhibited high cell toxicity and could not support enzymatic activity throughout the procedures. Then, in 1966 Norman E. Good and his team designed a series of buffers specifically for biological research with the following characteristics:

  • Buffers should have a pKa between 6.0 and 8.0 because the optimal pH for most biological reactions rests in this range.
  • Buffers should have high water solubility and minimum solubility in organic solvents so it remains in the aqueous medium of the biological system.
  • Buffers should not permeate cell membranes. The buffer should not accumulate in cellular organelles. This may not apply to your specific experiment. Zwitterionic buffers do not permeate cell membranes.
  • Buffers should have minimal salt effects because ionic buffers can be problematic if the biological system being studied is negatively affected by salts.
  • The buffer’s concentration, temperature and ionic composition of the medium should have a minimal effect on buffering capability (pKa).
  • The formation of complexes between a metal ion and the buffer results in proton release, which affects the pH of the system and may have an adverse effect on experimental results. Thus, these ionic complexes should be soluble and their binding constant must be known. A buffer with a low metal-binding constant is suitable for the study of metal-dependent enzymatic reactions. If your experimental design requires the use of a metal, then you should choose a buffer that does not form a complex with that specific metal.
  • Buffers should be stable and resist enzymatic and nonenzymatic degradation. And they should not interfere with enzyme substrates or resemble them.
  • Buffers should not absorb light in the visible or ultraviolet regions of the spectrum to prevent interference in spectrophotometric assays.
  • Their preparation and purification should be easy and inexpensive

What are the most common buffers and how are they used?

The characteristics considered by Good and his team are a good starting point when choosing the buffer for your specific experiment. Here, we are including a Biological Buffer Selection Guide ( click here or scroll down for the PDF) containing a list of the most common biological buffers and the specific techniques and experiments they are used for. We are also including a description of their properties including pH, buffering range, metal binding capabilities, advantages and disadvantages and links to the protocols for the stock solutions. 

In addition, we are including a Guide ( click here or scroll down for the PDF) of the most commonly used buffers in the lab describing their pH and composition.

Additional resources:

User guide for GoldBio buffers

GoldBio buffer chart, by family

GoldBio buffer chart, by pH


References

10X Running buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10475.full?text_only=true.

Ferreira, C. M., Pinto, I. S., Soares, E. V., & Soares, H. M. (2015). (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review. RSC Advances, 5(39), 30989-31003. doi:10.1039/c4ra15453c.

Good, N., & Izawa, S. (1972). [3] Hydrogen ion buffers. Methods in Enzymology Photosynthesis and Nitrogen Fixation Part B, 53-68. doi:10.1016/0076-6879(72)24054-x.

Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., & Singh, R. M. (1966). Hydrogen Ion Buffers for Biological Research*. Biochemistry, 5(2), 467-477. doi:10.1021/bi00866a011.

Laboratory Stock Solutions and Equipment. (1998). Current Protocols in Cell Biology,00(1). doi:10.1002/0471143030.cba02as00.

Purich, D. L. (2010). Factors Influencing Enzyme Activity. Enzyme Kinetics: Catalysis & Control, 379-484. doi:10.1016/b978-0-12-380924-7.10007-9.

Tris-Glycine/MeOH Transfer Buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2015/5/pdb.rec087064.full?sid=3b0abcbe-5bcf-460f-87d8-48a5525a9d25.

SDS-PAGE Running Buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2014/7/pdb.rec081117.full?sid=f680f5fc-664e-4fe1-a840-07cec720d52e.

Zbacnik, T. J., Holcomb, R. E., Katayama, D. S., Murphy, B. M., Payne, R. W., Coccaro, R. C., . . . Manning, M. C. (2017). Role of Buffers in Protein Formulations. Journal of Pharmaceutical Sciences, 106(3), 713-733. doi:10.1016/j.xphs.2016.11.014.


Category Code: 79105, 79104, 88251


Biological Buffer Selection Guide



Guide: Commonly used Buffers

Posted by Karen on October 2nd, 2018  ⟩  0 comments

Blue jeans have an inseparable one-way word association, and what makes that so interesting is that blue jeans start out yellow – well, sort of.

When “jeans” are brought up with no other descriptor, the assumption is that a person is talking about comfortable blue pants. Jeans are warm, durable, relaxed, and cut in a variety of perfect fits. To top it off, their blue coloring seems to coordinate with pretty much any other color.

The jeans we wear today are the result of amazing evolutions in textile production and dye process. The history of jeans begins in Genoa, Italy and Nimes, France several centuries ago. These two locations, where the woven material was primarily produced and mastered, are where we get the terms jeans, from Genoa and denim, de Nimes.

Both places produced heavy cotton blends called fustian, and they used a durable diagonal weave style called twill. The twill woven cotton was ideal for working class and common people of the time since the material was relatively affordable, comfortable (jeans being the more comfortable of the two), and held up in more laborious settings.

By the mid-16th century, Genoa began having more success in exporting their fustian fabric to England, and later the American Colonies.

The color blue and its association to jeans is another element to the history, and one where some science is involved. Blue coloring can come from a number of dyestuffs, but indigo for textile dyeing offered many benefits.

Since ancient times, indigo dye has been preferred for its overall color fastness – its resistance to fading over multiple washes and exposure to light. The trouble with indigo is that it is not water-soluble, which means it will not naturally dye the intended fiber. This characteristic and its overall dye process makes indigo fall under the category of vat dye.

In order to make vat dyes work, dipped material must go through a reduction and oxidation stage, sometimes a series of them. During reduction, indigo dye is converted to its yellowish-green, water-soluble leuco-indigo form when in an alkaline solution with reducing agents. It is during this process that jeans start out yellow (or the yarn fibers do, depending on manufacturing process). After dipping, the fiber goes through oxidation, usually by air, which returns indigo to its fixed, water-insoluble blue. Multiple dips help increase the brilliance and darkness of the blue dye.

Another factor greatly contributing to color output is the pH of the dye bath. Vat dyes work well in alkaline conditions where fiber better absorbs the dye. For cotton blends, a pH of 11.5-12 is ideal for color absorption. In fact, pH has been such an important element in dyeing, that efforts to optimize the process have been introduced over the years. Something as simple as introducing fiber to the dye bath could change the overall pH of the solution just enough to decrease the efficiency of the dye. To stay within a narrow pH range, buffers, bases or acids enable a more effective dye process.

By the 19th century, the millennia of dye perfection and the centuries of textile perfection met at a perfect time in American history when gold prospectors were heading west and industrial production was in a golden age. It is during this time where Levi Strauss, a Bavarian immigrant, moved from New York to San Francisco to open a dry goods business. He catered to customers who not only needed dry goods, but also durable supplies, including good working clothes. In 1872, Strauss was approached by Jacob Davis, a tailor from Reno, Nev. Davis introduced a way of making jean pants that were more durable due to his design involving rivets around strain points of the pants. Strauss and Davis partnered together, patenting the design and marketing the pants. At the time, Levi jean pants came in two color options: cotton duck (like canvas), and blue denim.

How blue outlived the cotton duck goes right back to the science of indigo dye. Each wash would strip away tiny amounts of the indigo dye. But because the dye had tightly bonded to the fiber, each wash also meant that little threads of the cloth were stripped with it. Every wash softened the material, making it more and more comfortable. The fading quality of the indigo dye contributed to a rugged look and an unwritten personal story. By 1911, Strauss phased out the duck color and ran only with blue.

Blue jeans began to symbolize ideals of the American West. By the 1950s, partly thanks to Hollywood, jeans became associated with rebellion, youth and freedom. Since then, jeans have become one of the most common, universal garment.

The history and science behind blue jeans goes way deeper than this overview. A great read is “The Master of the Blue Jeans,” a book that explores an unnamed artist of the late 17th century. The artist is dubbed the Master of Blue Jeans because many subjects are depicted wearing garments made of blue denim. Find the book online here: https://issuu.com/artsolution/docs/cat._maitre_toile_de_jeans_a

References

Adnan(2010, January 14). Dyes Used For Denim Dyeing – A Description. Retrieved September 17, 2018, from https://www.denimsandjeans.com/denim/manufacturing-process/dyes-used-for-denim-dyeing-a-description/1397

Chakraborty, J. (2003, June 30). Dyeing of denim with indigo. Retrieved September, 2018, from http://www.niscair.res.in/sciencecommunication/ResearchJournals/rejour/ijftr/Fulltextsearch/2004/March 2004/IJFTR-Vol 29-March 2004-pp 100-109.htm

Driessen, K. (n.d.). The Earliest Dyes. Retrieved September, 2018, from http://www.quilthistory.com/dye.htm

Dyeing Process | Different Types of Dyes | Classification of Dyes. (n.d.). Retrieved September 12, 2018, from http://textilelearner.blogspot.com/2011/07/dyeing-process-different-types-of-dye_1720.html

Gruber, G., Canesso, M., Girbaud, F., Frangi, F., Morandotti, A., & Cataldi Gallo, M. (2010). The Master of the Blue Jeans - Paris Galerie. Paris: Galerie Canesso. doi:ISBN 978-2-9529848-4-3

Hegarty, S. (2012, February 28). How jeans conquered the world. BBC.

History of Dungaree Fabric. (n.d.). Retrieved September, 2018, from http://www.historyofjeans.com/jeans-history/history-of-dungaree-fabric/

Rajveer. (n.d.). Alkali Buffers (Soda Ash Replacements). Retrieved September, 2018, from http://www.foodcolorworld.com/alkali-buffers.html

Rippon, J., Cai, J., & Smith, S. (n.d.). Dyeing Wool with Metal-free Dyes – The Use of Sodium Borohydride for the Application of Vat Dyes to Wool. Retrieved September, 2018, from https://publications.csiro.au/rpr/download?pid=csiro:EP112736&dsid=DS4

Stewart, J. (2013, October 14). Why Are Jeans Blue? Retrieved September 12, 2018, from http://www.slate.com/blogs/the_eye/2013/10/14/blue_jeans_what_s_the_reason_behind_the_color.html

Wolff, L. (2011). What's That Stuff? Blue Jeans. Science and Technology,89(43). Retrieved September 17, 2018, from https://pubs.acs.org/cen/science/89/8943sci3.html.


    
              Karen Martin
GoldBio Marketing Director


"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: 79101

Posted by Karen on September 11th, 2018  ⟩  0 comments

You’re planning a birthday party, something amazing for someone amazing, and you want it to be memorable. So you hire a great DJ and book a swank venue, but you’re looking for one more thing to set it apart, and then it hits you – glow-in-the-dark food and beverages! You start the hunt for recipes and tips. Popping open glow sticks and mixing them in cake frosting is not an option since glow sticks are toxic. But in your quest, you start seeing the word “bioluminescence” come up, and wonder if that might solve the problem.

Make glowing drinks

This is actually a question that GoldBio has been asked many times. People wonder if they could buy luciferin and mix it in food and drinks to make them glow.

It ’s a cool idea! But, unfortunately, the answer is no. Luciferin is not the way to go about this.

First, GoldBio products are not for human or animal consumption. Our products should not be orally ingested or administered as food, medicine or supplements. GoldBio products are for research purposes only.

Second, the way the luciferin-luciferase chemical reaction works causes another obstacle in your party planning. Making food glow with luciferin alone will not work. The substrate requires the luciferase enzyme along with other reagents in specific conditions.

There was a kit called Food Magic. It incorporated the mechanics of bioluminescence into one of its recipes. Food Magic came with instructions and supplies for making bioluminescent syrup. Unfortunately, Food Magic does not appear to be available to the market anymore.

Another company, Biolume, tried to commercialize the entertainment aspects of bioluminescence. The company trademarked the brand Lumoness. According to their website, their goal is to “illuminate a new segment in the food, beverage, cosmetic and medical imaging markets with natural bioluminescence.” It is unclear how far they have gotten with this mission since there are no products listed, and searching for Lumoness food or cosmetics leads to unrelated results.

So currently, commercializing luciferin or coelenterazine for food and beverages has led to a dead end.

In doing further research on bioluminescent food, there was some hope when discovering that Japan has a delicacy called firefly squid. The squid produces a beautiful, bioluminescent, blue glow, and is delicious. But they lack their glowing abilities once they’re boiled or fried and ready for eating.

Even though bioluminescence doesn’t offer much hope when planning an unforgettable event, you're not out of luck. There is another option to consider, fluorescence. Fluorescence occurs when an object containing phosphors has absorbed light and then emits light. It differs from bioluminescence, which uses a chemical reaction to produce light. One way to achieve fluorescence is with UV light (black light).

So what offers the ability to fluoresce and is safe for food? Tonic water. Tonic water contains the chemical quinine that fluoresces under black light. There are still some minor safety considerations when using tonic water: Anyone who suffers abnormal heart beat, has low blood sugar, is pregnant, has kidney or liver disease or is taking medications should avoid tonic water.

Check out this recipe from instructables.com for glow-in-the-dark punch using tonic water and black lights. Instructables.com also has a cool glow-in-the-dark Jell-O recipe.

A helpful tip when exploring or experimenting with your own tonic water recipes is that the more opaque the primary ingredients are the less effect tonic water will have under a black light. There is also fear that tonic water might negatively alter the flavor of your food or drinks. Oftentimes, sugar is the best way to keep things tasting good. There are many other recipes online that you can find to give you more tips on how to balance the ingredients so that everything tastes great.

Tonic water and black lights might not be quite the solution you were looking for, but it is safe, cost-effective, has a cool effect, is very easy to work with and gives you the ability to experiment and customize.

If you’re interested in exploring other things that glow under black light that might lend to your party planning, here are a few web articles that might be helpful:

But please…don’t consume the luciferin.

References:

Betton, P. (2011, November 06). Host a glow-in-the-dark party. Retrieved August 20, 2018, from https://www.telegraph.co.uk/lifestyle/8870854/Host-a-glow-in-the-dark-party.html

Biolume Home. (n.d.). Retrieved August 21, 2018, from http://biolume.net/

Helmenstine, A. (2017, October 31). How To Make Glow in the Dark Face Paint. Retrieved August 21, 2018, from https://sciencenotes.org/how-to-make-glow-in-the-dark-face-paint/

Helmenstine, A. M. (2018, July 10). 16 Things That Glow Under Black or Ultraviolet Light. Retrieved August 20, 2018, from https://www.thoughtco.com/what-glows-under-a-black-light-607615

Ijmiers1. (2017, October 19). Glow in the Dark Jello! Retrieved August 21, 2018, from https://www.instructables.com/id/Glow-in-the-Dark-Jello/

Sykes, T. (2011, March 7). Fancy a Pancake that Glows in the Dark? The Willy Wonka Kit that Adds Magic to Meals. Retrieved August 17, 2018, from http://www.dailymail.co.uk/news/article-1361336/Heston-Blumenthal-inspired-kitchen-chemistry-kit-glow-dark-pancake-recipe.html

Travowolf. (2017, October 19). How to Make Glow-In-The Dark Punch for Halloween. Retrieved August 21, 2018, from https://www.instructables.com/id/How-to-make-glow-in-the-dark-punch-for-Halloween/

Web Japan. (n.d.). Toyama: Firefly Squid. Retrieved August 20, 2018, from https://web-japan.org/kidsweb/local/firefly-squid/index.html


    
              Karen Martin
GoldBio Marketing Director


"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: 79101

Posted by Megan on September 4th, 2018  ⟩  0 comments

It was August when thousands of bikers assembled in a city street, the dark evening ignited by streetlamps and wheels aglow with hues of red, green, blue and purple. A radiating energy pulsed through the crowd as gears shifted and feet were poised over pedals. The sound grew until a cheer of excitement rumbled, echoing between the buildings and towards the stars above. With a signal given, the bikers all took off at once and carried with them a hypnotic technicolor display of reflectors, string lights, and the ever-popular luminescence of glow sticks.

glow stick science and history

This is the Moonlight Ramble, a semi-annual bike ride through the roads of St. Louis, Mo. Participants cover their bikes in fluorescent rainbows, braiding them through the spokes and adorning their handlebars with vibrant color to match the city. The ghostly kaleidoscope sweeps through St. Louis like wil-o-wisps or clouds of fireflies; the effect is spellbinding and evokes the summer season.

One of the main attractions for events like the Moonlight Ramble is the display of city lights and decorative elements like glow sticks. Glow sticks are popularly used to brighten concert, party, and camping venues, always providing a fascinating source of light and color.

Glow sticks are one example of luminescence, the same principle which gives fireflies the power to light up and promotes the glow of some biochemical experiments. Such bioluminescent processes require luciferin or related bioluminescent substances; although glow sticks don’t contain luciferase reactions, they are no more complicated than the simplest radiant assay.

To honor luminescent sciences and end-of-summer festivities, we will illuminate the history behind these mesmerizing party favors, explain their mechanism, and suggest some experiments you can conduct with them.

Invention

From bike decorations to Halloween accessories, glow sticks have wide recreational use, but they were originally patented as a signaling device. This function is different from molecular chemical signaling – glow sticks were intended as emergency flares minus the open flame. Their reliable light source could be deployed in an emergency or by first responder services. Military personnel, divers, police and fire fighters were equipped with glow sticks to illuminate their fields of operation.

With this original purpose expanded, we now see the same devices being used at concerts and worn by costumed elementary schoolers. Even the inventor of the contemporary glow stick, Dr. Edwin Chandross, was surprised by their recreational popularity. When told about their use in music venues, his response was "Is that so? Maybe my granddaughter will think I'm cool now."

Dr. Chandross was inspired by witnessing luminol chemiluminescence at the Massachusetts Institute of Technology. He learned peroxalate esters are chemiluminescent when reacting with hydrogen peroxide; his discovery of the ideal proportion took one day, but he never patented it. Several years later, glow sticks using his formula were manufactured and sold to the public.

Mass-produced glow sticks were first applied in military activities then repurposed for concert apparel. Vice has published lore from the music community, citing the first instance of this practice at a 1971 Grateful Dead concert in New Haven, Conn. Here, the crowd began throwing activated glow sticks on stage for the performers to hold. This event became a trend that is now a tradition of widely distributing glow sticks at music performances worldwide.

Glow Stick Science

Glow sticks have a high value when it comes to low-cost entertainment: no batteries or electricity necessary, cheap to produce, portability and high functionality as well as resilience. They last for hours and cost dollars for dozens of tubes. They’re the perfect party favor or location marker just as bioluminescent indicators illuminate our experiments and track important molecules.

The shine of these tubes is engendered by chemiluminescence rather than bioluminescence. This reaction is not organic, yet when chemical substances are combined, they produce an equally vibrant glow without additional sources of energy. In chemiluminescence, electrons in chemical compounds are excited, and their return to a normal level releases energy as light.

It only takes the mixing of a few chemicals to produce the hypnotic glow. A base, often sodium salicylate, catalyzes this reaction with a dye to create an exothermic reaction. These chemicals, along with diphenyl oxalate, compose the outer solution within the glow stick tube. An inner glass vial holds hydrogen peroxide, and when this is mixed with the phenyl oxalate ester, phenol and peroxyacid ester are produced.

Peroxyacid spontaneously decomposes to activate the fluorescent glow stick dye with its energy. The dye is then responsible for releasing photons when its electrons are excited. The faster these chemicals mix, the faster and more luminescent the reaction. This is why breaking the glow stick in multiple places and shaking it results in a brighter output.


Glow stick science - how a glow stick works


The concentrations of substances in this solution can be adjusted for a shorter, brighter glow or a lasting, dim light. Activation levels of light are the highest in energy and decay exponentially unless specific procedures are undertaken to revise this. By manipulating concentration, certain chemiluminescent recipes can engender brilliant light in a flash rather than glow. High quantities of sodium salicyate or other bases can have this effect.

How do glow sticks generate different colors, then? The incorporated dye determines photon wavelength, visible as the color of light emitted. Light is the most prevalent energy product of the reaction, because chemical transformations occurring within the tube can’t be sustained by a thermal reaction.


Glow Stick Dye Chemistry


As such, glow sticks can be specifically designed to function in hot or cold climates. Heating a glow stick – like adding base substances – will encourage a faster, brighter, briefer light omission. Don’t microwave a glow stick or place it in/on any heating device to see it brighten; this is hazardous to you and your appliance because the tube can burst and spill fluid and glass everywhere. Alternatively, cooling extends the reaction with less extreme glow. An activated glow stick frozen or refrigerated will dim but resume glowing when the temperature rises again. The cold augments glow longevity. Glow stick dyes also retain their fluorescence under ultraviolet light – even a spent glow stick may brighten when exposed to black light.

For their economical cost and simplicity, these handheld science experiments are surprisingly versatile. Glow sticks can also be manufactured at home or in a laboratory. We’ll demonstrate some of their uses by outlining easy experiments anyone can conduct.

Home and Laboratory Experiments

Glow sticks can be manipulated with temperature, and the following experiment optimizes this property while maintaining the simplicity of glow sticks. The materials you need include a thermometer, two cups (preferably glass), water and ice, temperature-regulating gloves, tongs and three glow sticks of the same color.

Fill the cups, one with hot or near boiling water and the other with ice water. Snap and activate each glow stick, shaking them to start the reaction within. With the tongs, place one glow stick in each of the cups and leave the third out – this is the control, “room temperature” example. For the next several minutes, observe the varying reactions of each glow stick. You’ll notice the luminescence from the heated glow stick is brighter, but its light will be maintained for a shorter time; meanwhile, the near-frozen glow stick will have a dimmer glow but last longer.

An alternative method with this experiment would be to activate two glow sticks in the morning, storing one in the pantry and another in the freezer for the day. If they’re compared at night, the glow stick which was frozen should be more resilient.

For more involved fluorescent science, there are experiments for the laboratory environment. This involves more of a cautious approach, but it has a satisfying conclusion. The supplies include: six containers (again, glass), gloves and goggles, a tray or other platform, paper towels, a colander, an incising tool and three large, colored glow sticks: yellow, blue and pink. For a more robust sample, add extra glow sticks of the same colors.

Divide the containers into pairs. Put on the gloves and goggles before carefully cutting the top of each glow stick open and pouring the contents into individual glasses. All substances in the experiment are non-toxic, but the inner glass vial should be cautiously removed, rinsed (to prevent a precocious reaction), and broken open into the second set of containers. Avoid losing glass in the solution using the colander.

The pairs of containers should not be reacting, and turning off the lights in the room should demonstrate this – there is no glow. To initiate the reaction, add the activator (liquid from the glass vial) into the dye for each pairing. It should glow almost immediately!

To see an even more impressive reaction, the three reacting solutions can be poured together to produce a singular white glow. This result is accomplished through the combination of all light frequencies represented by the three glow stick colors. Dispose of the glass and liquid with the same protocol as other biochemical products.

Making Your Own Glow Sticks

We’ve covered the invention, science and applications of glow sticks, but what about their production? If you’re interested in creating your own fluorescent solution, we have a few recipes for you. There are potentially hazardous materials involved, so we recommend using gloves, a facemask and goggles in a well-ventilated setting. It is not recommended that children help with this experiment.

The chemical ingredients include hydrogen peroxide, sodium peroxide, potassium ferricyanide (the catalyst), luminol and water. Other supplies: a glass container and stirring instrument.

First, create the base inside the glass container. This solution consists of 1.5ml of hydrogen peroxide, .04g sodium hydroxide, .04g of luminol and 40ml of water. The solution should be thoroughly mixed with the stirring instrument to dissolving. To activate your base, slowly pour in potassium ferricyanide; higher quantities of this chemical will elongate the glow. It’s that simple!

This solution can be revised with a well-mixed base of 10ml diethyl phthalate, 100mg sodium acetate and 50mg TCPO. Add 3g of your choice of fluorescent dye for color. More TCPO and sodium acetate in the solution will increase light output. Combine the catalyst for this recipe, 3ml of 30% hydrogen peroxide. Now, you have an authentic chemiluminescent glow!

A long-lasting, solar-powered glow stick is also possible. Zinc sulfide – a glowing powder – can be combined with epoxy resin and hardened in a tube to produce a permanent glow stick, similar in operation to the glowing star stickers kids put on walls and ceilings. With sunlight exposure, the zinc sulfide absorbs and projects luminescence in the dark. This is a reusable option that provides endless light.

We hope you enjoy the simplicity of luminescence, whether you pick up a box of glow sticks from the store or craft your own in the laboratory!

References

Gaston, B. The Guy Who Invented Glow Sticks Had No Idea They Were So Popular (2013, November 26). Retrieved August 2, 2018, from https://thump.vice.com/en_us/article/4x8gjn/the-guy-who-invented-glow-sticks-had-no-idea-they-were-so-popular.

Glow Stick Science: Chemical Reaction Lab. Retrieved August 2, 2019, from https://www.adabofgluewilldo.com/glow-stick-science-chemical-reaction-lab/.

Harris, T. How Light Sticks Work (2001, November 1). Retrieved August 1, 2018, from https://science.howstuffworks.com/innovation/everyday-innovations/light-stick.html.

Heinecke, L. Glow Stick Science Experiment for Kids (2018, February 9). Retrieved August 2, 2019, from https://www.scholastic.com/parents/school-success/learning-toolkit-blog/glow-stick-science-experiment-kids.html.

Nichols, M. How to Make Glow Sticks Yourself — Backed by Science! (2017, January 3). Retrieved August 2, 2018 from https://schooledbyscience.com/make-glow-sticks-backed-science/.


Megan Hardie is an undergraduate student at The Ohio State
University’s Honors Arts and Sciences program. Her eclectic
interests have led to a rather unwieldly degree title: BS in
Anthropological Sciences and BA English Creative Writing,
Forensics Minor. She aspires to a PhD in Forensic Anthropology
and MA in English. In her career, she endeavors to apply the
qualities of literature to the scientific mode and vice versa,
integrating analysis with artistic expression.
          Megan Hardie
      GoldBio Staff Writer
Posted by Karen on August 14th, 2018  ⟩  0 comments

Making sure your experiment goes right is a top priority because it saves time, money and prevents the overall frustration of the job. In many DNA extraction protocols, the use of proteinase K is an important step because of its ability to digest harmful nucleases, but how much to use, when to use it and for how long can sometimes be a mystery. In this article, we untangle 5 common proteinase K questions that relate closely to extraction methods. While we hope that this article serves as a helpful guide in your work, it is critical to do additional research to make sure your methods are perfectly matched to the type of work you’re doing.

To view a printable proteinase K digestion table, click here or scroll down for the PDF.

Proteinase K Protocol and Digestion Guide



When is proteinase K used?

Proteinase K is used mostly in DNA and RNA extraction protocols. You’ll often find the proteinase K step within the lysis section of the protocol. For example, in the nucleic acid extraction protocol, proteinase K is added to cell lysate and then an incubation period follows to ensure a complete digestion.

To prevent potential digestion of your samples, proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C.

Even in the typical mouse-tail protocol, proteinase K is regularly used to inhibit harmful nucleases. And the addition of proteinase K occurs during the digestion step. The use of EDTA is also suggested to help the inactivation of nucleases by inhibiting Mg2+ dependent nucleases.



How do I know if digestion has happened?

Usually, the biggest tell that complete digestion has occurred is that you should see a clear lysed cell solution. If you are not seeing a clear solution after the initial digestion period, extend your incubation time.

Be very careful with this. If you are using a faster method for isolation, especially involving higher volumes of proteinase K, you’ll need to pay close attention during proteinase K digestion. A longer digestion may cause degradation of your DNA.



How long of an incubation time should I use?

The incubation period with proteinase K is going to depend primarily on the type of sample you’re working with. After doing quite a bit of research, here is the range of times we found for different cell and tissue samples. Please keep in mind that your experiment may have different requirements or variables that could greatly influence digestion times, and therefore we strongly encourage you to do your own research before carrying out your work.

Formalin-Fixed Paraffin-Embedded Tissues Digest for several hours to overnight.

*Note: Formalin-Fixed Paraffin-Embedded Tissues commonly appears in the abbreviated form, FFPE. This is a method for tissue preservation (another long-term tissue preservation method is with frozen tissue).

  • Bacteria – Digest with proteinase K between 1-3 hours. Digestion temperature may also influence how long your digestion should take.
  • Mammalian cells – There are several papers out there with a wide range of stated digestion times – as little as 1 hour and as long as twelve hours. This is partly due to experimental objectives and the type of cells used. Digestion temperature and proteinase K volumes also have some influence.



What temperature should I use for proteinase K digestion?

Digestion temperatures also vary with the type of sample you’re working with. Once again, we offer a guide on this, but strongly encourage you to do more research to optimize all conditions before proceeding with your experiment.

  • FFPE Tissue – Digestion temperatures 55-56°C. Most articles are fairly consistent with that temperature range.
  • Bacteria – Digestions are often carried out at 55°C. Some articles, however, did state a 37°C digestion temperature. Keep in mind the requirements of the type of sample you’re working with and other factors of your experiment.
  • Mammalian – Articles greatly varied in digestion temperatures. Shorter digestion periods usually correlated with higher temperatures (optimal proteinase K digestion temperatures for mammalian cells range between 50-65°C). Articles with digestions taking place for several hours to overnight usually suggested 37°C. Several other factors can impact the digestion temperature such as cell type (blood, buccal, etc.) and molecular weight.



How much proteinase K do I use?

The amount of proteinase K you need for successful digestion is going to depend on many factors: the protocol you’re using, the type of sample you’re working with, the conditions of your experiment, etc. Typically, 10-20 µl of proteinase K are used in experiments, with stock proteinase k stock concentrations usually around 20 mg/ml.

Something else to keep in mind is that some methods require a second digestion step (usually those involving tissue samples). Weaker, second digestions usually call for a lower volume and a different digestion period.

For more proteinase K tips, visit the product page for a list of related literature, or check out our articles on common questions about proteinase K and proteinase K activity.



References


Bielawski, K., Zaczek, A., Lisowska, U., Dybikowska, A., Kowalska, A., & Falkiewicz, B. (2001). The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis. International Journal of Molecular Medicine.doi:10.3892/ijmm.8.5.573Biase, F. H., Franco, M. M., Goulart, L. R., & Antunes, R. C. (2002). Protocol for extraction of genomic DNA from swine solid tissues. Genetics and Molecular Biology,25(3), 313-315. doi:10.1590/s1415-47572002000300011

Chen, Z. (n.d.). Zhibin’s Protocol of Tissue Processing for PCR genotyping. Zhejiang University. Retrieved July 17, 2018, from http://person.zju.edu.cn/attachments/2012-05/07-13...

Derua, Y. A., Alifrangis, M., Hosea, K. M., Meyrowitsch, D. W., Magesa, S. M., Pedersen, E. M., & Simonsen, P. E. (2012). Change in composition of the Anopheles gambiae complex and its possible implications for the transmission of malaria and lymphatic filariasis in north-eastern Tanzania. Malaria Journal,11(1), 188. doi:10.1186/1475-2875-11-188

DNA EXRACTING FROM TOE PADS OR FEATHERS USING GENECLEAN II. (2005). Lougheed Genetics Laboratory Manual, Queens University. Retrieved July 17, 2018, from http://www.ispybio.com/search/protocols/dna_extractios_etc.pdf

DNA Isolation From Blood or Tissue Using Phenol/Chloroform. (2005). Lougheed Genetics Laboratory Manual, Queens University. Retrieved July 17, 2018, from http://www.ispybio.com/search/protocols/dna_extrac...

Fan, H., & Gulley, M. L. (2001). DNA Extraction from Fresh or Frozen Tissues. Molecular Pathology Protocols,5-10. doi:10.1385/1-59259-081-0:5

Feil, W., Feil, H., & Copeland, A. (2012, November 12). Bacterial genomic DNA isolation using CTAB [Web log post]. Retrieved July 16, 2018, from https://www.researchgate.net/profile/Ali_Mahmoudpo...

Goldenberger, D., Perschil, I., Ritzler, M., & Altwegg, M. (1995). A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Research,4(6), 368-370. doi:10.1101/gr.4.6.368

Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., & Lange, T. D. (1999). Mammalian Telomeres End in a Large Duplex Loop. Cell,97(4), 503-514. doi:10.1016/s0092-8674(00)80760-6

Gross-Bellard, M., Oudet, P., & Chambon, P. (1973). Isolation of High-Molecular-Weight DNA from Mammalian Cells. European Journal of Biochemistry,36(1), 32-38. doi:10.1111/j.1432-1033.1973.tb02881.x

Hrncirova, K., Lengerova, M., Kocmanova, I., Racil, Z., Volfova, P., Palousova, D., . . . Mayer, J. (2010). Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis. Journal of Clinical Microbiology,48(9), 3392-3394. doi:10.1128/jcm.01109-10

Lum, A., & Marchand, L. (1998). A Simple Mouthwash Method for Obtaining Genomic DNA in Molecular Epidemiological Studies. Cancer Epidemiology, Biomarkers & Prevention,7, 719-724. Retrieved July 17, 2018, from http://cebp.aacrjournals.org/content/cebp/7/8/719....

Meulenbelt, I., Droog, S., Trommelen, G., Boomsma, D., & Slagboom, P. (1995). High-Yield Noninvasive Human Genomic DNA Isolation Method for Genetic Studies in Geographically Dispersed Families and Populations. The American Society of Human Genetics.,1252-1254. doi:0002-9297/95/5705-0038$02.00

Mirmomeni, M., Ma, S. S., Sisakhtnez, S., & Doranegard, F. (2010). Comparison of the Three Methods for DNA Extraction from Paraffin-Embedded Tissues. Journal of Biological Sciences,10(3), 261-266. doi:10.3923/jbs.2010.261.266

Parzer, S., & Mannhalter, C. (1991). A rapid method for the isolation of genomic DNA from citrated whole blood. Biochemical Journal,273(1), 229-231. doi:10.1042/bj2730229

Pikor, L. A., Enfield, K. S., Cameron, H., & Lam, W. L. (2011). DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses. Journal of Visualized Experiments,(49). doi:10.3791/2763

Rohland, N., & Hofreiter, M. (2007). Comparison and optimization of ancient DNA extraction. BioTechniques,42(3), 343-352. doi:10.2144/000112383

Sengüven, B., Baris, E., Oygur, T., & Berktas, M. (2014). Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues. International Journal of Medical Sciences,11(5), 494-499. doi:10.7150/ijms.8842

Shahriar, M., Haque, M. R., Kabir, S., Dewan, I., & Bhuyian, M. A. (2011). Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains. Stamford Journal of Pharmaceutical Sciences,4(1). doi:10.3329/sjps.v4i1.8867

Steinau, M., Patel, S. S., & Unger, E. R. (2011). Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues. The Journal of Molecular Diagnostics,13(4), 377-381. doi:10.1016/j.jmoldx.2011.03.007

Wilson, K. (2001). Preparation of Genomic DNA from Bacteria. Current Protocols in Molecular Biology,56(1). doi:10.1002/0471142727.mb0204s56

Zoetendal, E. G., Ben-Amor, K., Akkermans, A. D., Abee, T., & Vos, W. M. (2001). DNA Isolation Protocols Affect the Detection Limit of PCR Approaches of Bacteria in Samples from the HumanGastrointestinal Tract. Systematic and Applied Microbiology,24(3), 405-410. doi:10.1078/0723-2020-00060




    
              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. 




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Proteinase K Protocol Digestion Guide

This guide shows the experimental conditions for digestion with proteinase K.