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Posted by Megan on August 1st, 2018  ⟩  0 comments

The traveling salesman, 45 and otherwise healthy, had been hospitalized several times already.

Based on a real homicide case.

The traveling salesman, 45 and otherwise healthy, had been hospitalized several times already. He complained of fatigue and weight loss, a condition that had persisted for half a year. Doctors hadn’t come to any diagnosis; they weren’t even sure if he had a disease, because no tests they’d conducted offered conclusive results. His main physician referred the salesman – we’ll call him Frank – to a pathologist, a doctor who often performs disease analysis in a forensic context. That pathologist, Dr. Daniel, asked for a sample from the patient that Frank’s physician may have thought unusual: fingernail clippings.

This story might be resemble the beginning of a medical or crime drama many of us are familiar with. However, much of the forensic science presented in crime shows is actually conducted in real labs. When a crime like robbery or murder is committed, scientists are consulted to help identify a perpetrator, and they often rely on DNA evidence from body fluids like blood. But if there is no biological evidence left behind by a criminal – as in the case of poisoning – forensics experts have alternate methods of discerning what substance may have been responsible.

This situation usually requires a toxicologist, an expert on chemical analysis and substance reactions, whose knowledge can identify what poisons are present in a body or tissue. The sources they collect may be blood or vitreous fluid from the eyes; they also sample nails or hair. Toxicological analysis can improve law enforcement’s chance of solving a crime if one exists, providing investigators with biochemical and DNA profiles. While Frank’s identity was known, his fingernail samples still carried supplementary evidence pivotal to diagnosing his mysterious ailment.

Dr. Daniel understood the forensic value inherit to this man’s nails and their cellular composition. As a medical professional, he knew the dynamics of tissue growth: cells rely on and grow in the matrix of the body, but they incorporate environmental chemicals and substances as well. These molecules accumulate in the cellular complex, and they can be stored in the body for years. Such chemicals can be detected much later through biochemical tests. Nails in particular have a slow rate of growth, offering retrospective insight to the past year’s growth depending on the individual and length of the nail. Substances can be detected, identified and quantified through techniques developed by forensic specialists.

Frank’s nail clippings were easily packaged and transferred for additional analysis; only a small portion would be necessary for the process. Though Dr. Daniel had already made a confident visual analysis on the nails, he wanted a second opinion, and the added confirmation would hold weight in a court system he was now certain the case was destined for. Ancillary tests had to be conducted by the toxicologist who next received Frank’s fingernail clippings along with the chain of custody, a document passed between investigators to conserve the legitimacy of evidence. The toxicologist signed the form and started planning the best tests for her examination of the nails.

Nails and hair have special advantages in the forensics laboratory. The process of collecting these samples is simple, noninvasive and negligible in the physical integrity of a victim or perpetrator. Both sources are also robust in their resistance to chemical change – the durability and slower decomposition rate of keratin preserves nails and hair, even at room temperature. There are specialized chemicals used to counteract these properties, and these would be employed by the toxicologist.

A study published in the Journal of Analytical Toxicology confirmed certain substances, incorporated into the keratin fibers themselves, can be detected in nails for 3–6 months. This study of over 10,000 samples also indicated nails provide a longer potential timeline of exposure than hair does, raising their value for the forensic scientist. The toxicologist had to consider the options of tests she would prioritize to develop her results and the most valuable conclusions. There were a few different theories she could make based on what tests she performed. Nails as a substrate can diagnose general or environmental pathologies, indicating health concerns like infectious disease or trace elements from chemical exposure.

Initial tests performed include enzyme-linked immunosorbent assay and liquid chromatography–tandem mass spectrometry (LC–MS-MS). Samples that test as presumptive positive then go through additional confirmatory tests. In preparation, they are washed, pulverized and chemically digested. This process is another familiar method to biochemical laboratories, employing many of the same techniques and materials. Depending on the extraction performed, tests can provide scientists with the sample’s DNA profile or a summary of incorporated chemical components.

The priority of this case, the extraction of unknown substances from biological samples, is an important duty of forensic specialists and medical examiners. Chemical extraction was performed by the toxicologist analyzing Frank’s nails. A similar process is used by forensic scientists conducting DNA identification using the extraction of exogenous DNA.

Protocol by the Office of the Chief Medical Examiner in New York City entails the processing of these samples, applied when nails contain DNA or chemical information to medicolegal cases. Their keratin composition is first degraded by a digestion buffer; organic extraction involves a digestion buffer of Tris (pH 7.5), EDTA, 0.1% SDS and proteinase K. The proteinase K is directly accountable for breaking down the tough keratin and thus freeing other molecules for inspection. Once the nails are digested, they’re incubated and centrifuged. Subsequently recovered substances (DNA, trace element, toxin or biochemical) are purified, concentrated and extracted for identification. Regardless of whether nail samples are processed individually or as a group, soaking clippings in digestion buffer results in the highest recovery rates of significant biomolecules.

Biochemical extraction is used in cases of assaults with direct contact between victim and assailant. DNA from the victim can be separated from that of the perpetrator for positive identification. It can also identify the person from whom the nail originated. One nail clipping constitutes a robust sample for DNA, and medical examiners have confirmed the sources can remain viable for upwards to twelve years after removal from live or deceased individuals. In a 2005 research project by medical examiners in Italy, nail samples from exhumed and skeletonized individuals (deceased within the last twelve years) were processed with another proteinase K, SDS and DTT buffer. The extracted DNA was then amplified with polymerase chain reaction to help produce a successful sequence. Their procedures yielded DNA sufficient to identify each individual when soft and bone tissue were inconclusive as compared sources, proving just one clipping can provide the identity of a person.

Just one clipping also – in Frank’s mystery illness – increased the toxicologist’s success rate for detecting other biochemical substances. She used the same technique a medical examiner might when isolating DNA, using a similar proteinase K buffer. Extraction was optimized by the forensic toxicologist to indicate and classify any toxins. This technique, usually used for drug detection, was recommended by Dr. Daniel to identify something more sinister: poisons.

The nails were analyzed for metals, including mercury, arsenic and lead; arsenic was detected in all samples. Dr. Daniel confirmed these findings with his own – multiple bands of transverse true leukonychia or “Mee’s lines.” These transverse white bands on the nails are a physiological presentation indicative of arsenic poisoning, and they were present on Frank’s nails upon Dr. Daniel’s initial visual analysis.

The diagnoses of both toxicologist and pathologist had an unpredicted result: Frank’s primary physician recalled a similar patient. Another traveling salesman, of the same age and sales territory, had also reported weakness and weight loss before dying of unexplained causes. The cases were too similar to ignore, and Dr. Daniel asked for a comparative study between the two salesmen. When the first individual was exhumed again, arsenic was also detected in his samples. Consequently, a criminal investigation was launched under suspicion of deliberate poisoning.

Similar cases of attempted and successful poisoning have occurred for centuries, with arsenic becoming less popular once detection tests were developed in the 1830s. They do, however, resurface occasionally, as in the 1995 murder attempted by St. Louis native Jim Boley against his wife, a case that garnered national coverage. More recently, a Chesterfield, Mo. case of domestic homicide involved mineral water laced with arsenic – refer to the baffling 2004 death of John Mullen. This later crime was months in the casebook before arsenic was identified. The forensic technique is more refined in today’s laboratories, with regulated procedures and quantities of reagents like the instrumental keratin-cutter, proteinase K.

Outside the lab, investigators discovered both salesmen (now regarded as victims of poisoning) regularly ate at the same department store lunch counter while they traveled. They were also consistently served by the same waitress. Based on the average daily growth rate of nails (0.1-0.15 mm), law enforcement approximated the dates during which the salesmen had consumed arsenic. The Mee’s lines of the undigested nail samples were measured, and an estimate of distance between incidents was made to prove multiple arsenic-related events. Based on the quantity of Mee’s lines formed, Frank’s exposure was substantial, perhaps over half a dozen meals laced with the poison. They aligned with logs from his traveling schedules; the Mee’s lines from both men corresponded to dates when they’d dined at the lunch counter. Investigators had new reason to pursue this waitress as a suspect.

In the interim of their analysis, Frank died of health complications directly associated to the arsenic poisoning. The case had once again ascended in criminal court, now to murder. With the laboratory’s instrumental results and testimony from Dr. Daniel, the waitress was arrested and convicted of murder. The conviction of the salesmen’s murderer prevented any more unknowing victims from dining on what would be their last meal. It also reassured the forensic community that nails are essential sources of evidence for such cases.

All this pseudo-noir drama and real-life crime, resolved with the help of a buffer that has been a useful tool in biochemical research.

References

Conklin, E. E. (2005, January 19). Dead Reckoning. Riverfront Times. Retrieved July 9, 2018, from https://www.riverfronttimes.com/stlouis/dead-recko...

Daniel III, C. R., Piraccini, B. M., & Tosti, A. (2004). The nail and hair in forensic science. Journal of American Academic Dermatology, 50(2), 258-261. doi:doi:10.1016/j.jaad.2003.06.008

Foran, D., Hebda, L., & Doran, A. (2015). Trace DNA from Fingernails: Increasing the Success Rate of Widely Collected Forensic Evidence (Rep. No. 249534). Retrieved July 9, 2018.

Hebda, L. M., Doran, A. E., & Foran, D. R. (2014). Collecting and Analyzing DNA Evidence from Fingernails: A Comparative Study. Journal of Forensic Sciences, 59(5), 1343-1350. doi:10.1111/1556-4029.12465

NYC Office of Chief Medical Examiner. (2016, October 14). Forensic Biology Protocols for Forensic STR Analysis [Digital protocol transcript]. Office of Chief Medical Examiner, New York City.

Piccinini, A., et. al. (2006). Forensic DNA typing of human nails at various stages of decomposition. International Congress Series, 1288, 586-588. doi:10.1016/j.ics.2005.08.029

Shu, I., Jones, J., Jones, M., Lewis, D., & Negrusz, A. (2015). Detection of Drugs in Nails: Three Year Experience. Journal of Analytical Toxicology, 39(8), 624-628. doi:10.1093/jat/bkv067


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 Megan on June 13th, 2017  ⟩  0 comments

Every lab has unique procedures when caring for their biochemical materials, but most teams operate on similar fundamentals. These practical methods are shared among colleagues to minimize waste and maximize chemical potential, but we usually don't discuss why our guidelines work. This article will explore one such process of preservation - desiccation.

The major questions covered:

  • What is desiccation?
  • Do chemicals expire, and why?
  • What is necessary for desiccation?
  • How is desiccation performed in a laboratory?
  • What chemicals need to be desiccated?


Defining Desiccation

If you’ve ever spent too much time outside on a very hot or cold day with low humidity, you’ve probably experienced a form of desiccation.

Desiccation can be defined in a broad sense as the state of being very, very dry. This can be assessed in the physical properties of biology and chemistry.

Biologically, desiccation occurs when an organism loses a certain quantity of its retained water, as when plants are not properly watered or have too much light exposure. This change is usually detrimental. Different organisms have higher susceptibility to desiccation damage: snails, frogs and salamanders are just a few vertebrates which cannot survive extended periods of desiccation. One commonly referenced example is a snail doused in salt (NaCl). The Na+ and Cl- ions disrupt the balance of cell membranes, and the snail exudes high quantities of mucus to displace the salt. It does this with such vigor that the body dehydrates - essentially death by desiccation.

In chemical contexts, desiccation is commonly regarded as a method of preservation. DNA is often found in ancient, dry remains due to the deoxygenated tissue being preserved from putrefaction. Whereas DNA in decomposing cells might remain intact for only months in a degrading environment, DNA in tissue kept ideally deoxygenated or dry can remain intact for decades (and for mitochondrial DNA, centuries or millenia).

Desiccation is also a preserving method for laboratory-based compounds. Some substances are naturally dry; others are saturated. In both cases, desiccation methods can be used to deter chemical contamination and promote longevity.

Water in a substance can represent a problem on account of contamination or hydrate bonds that change chemical composition. A sample’s water content and thus composition will change based on humidity and temperature.



Shelf Life and Expiration

Desiccation is necessary to preserve the freshness and durability of certain chemical substances. Think of placing food in cold, dry places and even in the near vicinity of baking soda. This environment extends shelf life by reducing the amount of ambient moisture and bacteria. For chemicals, dehydrating their surroundings prevents them from “expiring,” or going bad, too quickly.

Standard shelf life can be defined by the limit of time during which the substance (if properly stored) experiences no chemical or physical changes. Alternatively, a chemical’s expiration date – affected by the same characteristics as shelf life – entails the period of time a standard is viable after its first use. This is usually shorter than shelf life, one year being the common maximum. For substances that require it, desiccation will extend shelf-life and provide stable conditions before the standard expires.

It is difficult to isolate one reason for the “expiration” of chemical substances and laboratory products. The relative stability of certain chemicals will make their period of usefulness shorter after they've been used. Transpiration losses from a container’s outlets and water affinity are responsible for how much moisture might be lost or gained in an exposed environment. Stability and transpiration losses help determine the shelf life of a standard chemical. The quantity of the substance kept together will also influence its longevity; smaller portions usually have a shorter shelf life.

Chemical substances do expire even if they are kept in the best conditions. Organic objects exposed to the elements lose their freshness, and compounds also break down. Their properties lose viability; reactions become less effective; experimental results are thus no longer accurate. In this sense, a chemical product will expire without being used, especially if left unprotected from destructive forces like humidity. To keep often expensive substances functional for longer, desiccation is a useful procedure.




Equipment: Desiccators versus Desiccants

Knowing the theory of desiccation is the first stage. The second is knowing how to perform it.  Desiccators and desiccants are two important components to keeping chemicals pristinely dry.

A desiccator is a container, usually made of glass or plastic, which maintains the dryness of chemical materials. They might be equipped with indicators to confirm humidity levels or suspended above a desiccant to improve performance. A vacuum desiccator has an additional vacuum to promote drying. Reduced pressure is ensured by sealing the desiccator. The use of a desiccator is generally favored to the addition of other chemical desiccants directly into a sample.

Desiccants are substances which attract and prevent the movement of water in the environment. These hygroscopic substances are often used to keep containers, fridges or testing equipment consistently desiccated. Some examples of useful desiccants include silica (used in the packets found in new clothes and dried fruits), activated charcoal, calcium chloride and calcium sulfate. In the case of the unfortunate snail, NaCl is the desiccant.

Three functional groups of drying agents are employed to prevent humidity-induced change. The agent can combine with water at varying rates depending on conditional temperature, like anhydrous sodium sulfate; others, including alkali metals, react irreversibly with water; the final option is a molecular sieve. A multitude of compounds can function as laboratory drying agents.

Desiccants can remove the moisture in solvents if a reaction is intolerant to water. They are also responsible for drying solids stored above or near them. If this method doesn’t work, vacuum-drying desiccators or molecular sieves are employed.

Meter indicators are used to determine whether desiccants have lost their functionality. Certain indicators will change colors, often from blue or white to pink, once the desiccant begins losing its effect. The desiccant must then be exchanged for a new one.



Laboratory Desiccation: Desiccators, Desiccants, and Freeze-Drying

Most desiccation is conducted by researchers and managers of lab material. Storage requires an intimate knowledge of each chemicals’ needs to promote accurate quality control and conserve resources stored in large quantities.

For laboratory work, desiccator- or desiccant-mediated preservation is employed for temperature-sensitive or decomposition-prone substances otherwise heated on a hot plate or in a microwave system. Proteins, enzymes, microorganisms and plasma are all heat-sensitive and need to be dried in an alternative way.

When the container is large enough to fit a sample currently in use, a desiccator is reliable for preserving dryness. Vacuum desiccators remove any previous moisture from a chemical. In other cases, the addition of desiccants keep substances properly dry.

Chemicals are stocked in boxes and bags of different sizes, so the number of desiccants used depends on the container's size and contents.

  • Standards that will be used in the short-term only need one desiccant.
  • Multiple desiccants are used if the product will be preserved, weighed and used for over a year.

Desiccants must be replaced. The longer the desiccants last, the better. One bag might provide moisture relief for three to six months depending on how it's sealed within the material, so expired desiccants should be removed from the shelf more often than expired chemicals.

  • For frequently used chemicals like TRIS and ampicillin, the containers of which are opened more often for use, two 8-oz desiccants will keep the chemical desiccated if stored in bulk.
  • A less frequented substance like penicillin might have three or four desiccants stored with it.
  • Other materials can get up to five desiccants for extended periods of shelf stasis.

Not all laboratories operate on the same formula of product-desiccant ratio, but the general rule of mutual linear increase will apply.

In addition, freeze-drying – also known as cryodessication or lyophilization – may be necessary for preservation. Freeze-dry techniques use dilute solutions to remove ice sublimes and volatile liquids from a biological or pharmaceutical substance. A freeze-drying unit may be implemented to perform this technique, freeing ice and moisture before removing it in vapor form.

Freeze-drying is a common practice for pharmaceutical and biochemical industries. Biopharmaceutical products include vaccines and Streptokinase. Probiotic powders are also produced by freeze-drying bacteria. Alternatively, a biochemical substance with low molecular weight may be freeze-dried to remove solvents when filtration membranes are inefficient.



Chemicals Susceptible to the Dangers of Moisture

What laboratory chemicals are more susceptible to the dangers of moisture? Desiccation is not essential for all substances, especially if they are already in liquid form. Reason would suggest that solids and crystalline structures are more vulnerable to the characteristic dampening of humidity. There are some cases in which liquids must be desiccated, and this can be achieved through distillation. Gases, too, can be desiccated with absorptive material.

What of the functional groups of laboratory substances? Antibiotics seem to be frequently registered. Penicillins and aminoglycosides are particularly common: ampicillin, carbenicillin, geneticin, hygromycin B and kanamycin monosulfate are all recommended to be stored desiccated at -20°C. So too is the glycopeptide antibiotic vanomycin hydrochloride. NTC, a streptothricin, is desiccated and stored at 4°C.

Likewise, some enzymes like D luciferin are better off desiccated. The reducing agent TCEP-HCl and some buffers – TRIS and MOPS – are, too. Another product is IPTG, which must also be protected from light. Even some dyes must have their moisture regulated.

It’s apparent that a large number of chemical substances must be kept desiccated. Because desiccated materials represent a breadth of laboratory chemicals, it’s essential for those involved in the storage and use of these products to be vigilant of manufacturer recommendations.

Preservation instructions should be listed on the label or supplier’s description of the product. If storage directions are overlooked, there is a potential for both the product and the research it’s used for to be squandered.



References

Desiccation. (2017, June 01). Retrieved June 08, 2017, from https://en.wikipedia.org/

Harris, T. (2002, August 22). How Freeze-Drying Works. Retrieved June 12, 2017, from http://science.howstuffworks.com/

Howard-Reed, C., Liu, Z., Cox, S., Samarov, D., Lebber, D., & Little, J. (2011). Assessing the shelf-life of a prototype reference material for product emissions testing. National Institute of Standards and Technology. Retrieved June 9, 2017, from https://www.nist.gov/publications/.

Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP): Laboratory Sample Preparation (Vol. 2). (2004). Retrieved June 12, 2017, from https://www.epa.gov

Perrin, D. D., Perrin, D. R., & Armarego, W. L. (1980). Purification of Laboratory Chemicals (2nd ed.). Oxford: Pergamon Press. Retrieved June 9, 2017, from https://erowid.org/.



             Megan Hardie
         GoldBio Staff Writer

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. 

Category Code: 79104, 79107, 79108, 79109, 88221, 88231

Posted by Karen on May 7th, 2015  ⟩  0 comments

One day your PCR works; then when you repeat it, you get no results, and when you try yet again, you get nonspecific binding. It’s these situations that drive you to superstitious rituals and prayers to the PCR gods for mercy. Unfortunately, divine forms of troubleshooting yield few results.

#PCRLuckyBuddies

For a veteran in life science research, PCR has become second nature; however, I have seen undergraduate and graduate students highly stressed out about PCR. I have heard them utter to one another, “I’m about to see if this works. Wish me luck.” I knew one undergraduate student who struggled for an entire semester trying to make PCR work only to end up switching majors. After investing years into scientific coursework and research, we don’t want it to come to that. Instead, we’re here to help.

Rather than having a huge troubleshooting article with every single PCR tip known to scientists, I’ll break this into a series: nonspecific binding, no results, smearing, weak results and contamination.

4 PCR Tips When Encountering Nonspecific Binding:


1.  Aliquot Aliquot Aliquot: If you remember the article about fridge & freezer organization, certain areas of your upright freezer have a greater risk of unintentionally exposing your reagents to freeze-thaws. Outside of the freezer, you also run the risk of contamination to your reagents that might degrade them. Simply put, protect your supplies. Aliquot DNTPs, primers, etc. Only move your working vials into easily accessible freezer boxes. Store the rest of your stock in a more protected area at -80 °C.

2.  Negative Controls: This is a must in every PCR setup. In fact, my mentor made it a practice to set up the first two tubes and last two tubes to be positive and negative controls. This let both he and I see if contamination was ever a reason for a bad PCR. And it let us see whether or not it occurred throughout the whole process.

3.  Increase Annealing Temp: By increasing the annealing temperature, you’re driving specificity. In general, you want to use an annealing temperature that is 5 °C lower than the Tm of your primers.

4.  Touch-down PCR: In this process, the first stages of PCR should have a high annealing temperature, even higher than the estimated Tm of your primers. Following cycles have incrementally lower annealing temperatures. This gradual adjustment stops when you have reached the calculated annealing temperature of your PCR primers. The tricky part will be deciding on the incremental decreases you want to use. With the higher annealing temperature being used at the beginning, the resulting sequence will be the most specific and able to out-compete nonspecific results.

Stay tuned for more PCR tips in the following article. We hope that some of these tips begin to help you attain the results you’re hoping for.


    
              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 79108

Posted by Karen on February 25th, 2015  ⟩  2 comments

There are a lot of products in the lab that do similar things but were developed for a specialized purpose. And sometimes you’re in a position where only the alternative is available, but you’re unsure whether you can trust it.

how to choose between similar reagents

Let’s take a look at a few of these products and see exactly what the difference is in order to determine which one is more appropriate, given a certain experiment.

1. Tris vs. Tris HCl

     vs.  

Both can be used for electrophoresis, but why would you choose one or the other? Tris is much less expensive compared to tris HCl; however, tris HCl is meant to simplify the buffer-making process.

On its own, tris is the basic component of the buffer, while the acidic component of the buffer would come from adding HCl. Rather than working with HCl or NaOH (to adjust the pH of tris HCl), tris and tris HCl can be blended together to reach the desired pH.

Making a tris buffer solution with tris-HCl also prevents overshoot, which occurs when too much acid is accidentally added, meaning NaOH will need to be added to correct the situation.

Verdict:
In the end, it’s handy to have both in the lab. But either or can be used, so long as you have the acid or base to properly adjust it.

2. XTT vs. MTT

          vs.      

Once again, the most obvious difference is price. XTT is priced higher than MTT, yet they are both very similar in application. So how do they differ? And what justifies the difference in price point?

XTT holds some significant advantages over MTT. For example, XTT is very sensitive and has a higher dynamic range. Reactions with XTT result in a soluble formazan dye. This means the final solubilizing step is eliminated, which is not the case when using MTT. Removing this step also reduces the risk of error, such as air bubbles from Triton X-100 or SDS. Lastly, OD reading can be immediately taken after incubation when using XTT, while MTT usually requires a longer incubation for the solubilization of precipitate.

Verdict:
Whether you choose XTT or MTT, the job will get done. Choosing between the two depends on your preference. If you want to eliminate steps and reduce the potential for error, then XTT is your guy. But if those advantages are not worth the additional cost, which is very significant, then MTT is a great alternative. Both are very reliable for evaluating cell populations.

3. DTT vs. β-mercaptoethanol vs. TCEP HCl

      vs.          vs.     

If you have worked with β-mercaptoethanol (βME) in the past, then you are too familiar with the stench. Even after you dispose of your gloves, the smell lingers in your nose for some time. Despite its unforgettable scent and toxicity, it is much less expensive than DTT or TCEP. Sometimes saving money is worth the obstacles; however, there are some preferred applications for each of the three.

TCEP is known for its stability and lack of odor. According to some posts on Research Gate, many researchers prefer to use it for storage. By only storing protein stock in TCEP, you use less and incur a lower expense. TCEP is also useful if you’re doing UV detection of protein in buffer. This is because TCEP absorbs less UV than the other two reagents.

During protein purification, βME or DTT are the popular choices. DTT is a strong reducer, 7-fold stronger than βME, and it doesn’t have the odor that comes with βME. On the other hand, βME is far less stable. It evaporates from solution which means its concentration in solution will decrease with time. And to maintain equilibrium, more βME is required; otherwise, proteins won’t be sufficiently reduced, causing electrophoretic bands to be fuzzy.

Ultimately, it’s a matter of preference between the two, however some researchers suggest there being a benefit to using DTT for protein purification and for using βME when purifying smaller molecules.

Verdict:
In the end, this is yet another series where all candidates should have a place in your lab. Though if you must boil it down to two, choose DTT and TCEP. Neither of the two products smell quite as bad, and GoldBio’s prices justify the subtle advantages.

4. DTT vs. DTE

    vs.  

With all of this talk about reducing agents in the previous example, you might now be wondering about DTT vs. DTE (Cleland’s Reagents). This is one case where it’s simply a matter of preference and price. In Cleland’s original article, he stated that there appears to be no significant difference. They are epimers: the hydroxyl groups of DTE are in the cis form, while in DTT, they’re in the trans form.

Verdict:
Either or is fine. Go with the more cost effective product or the product you have the most experience with. At least now you know that if you’re ever in a situation where you must “borrow a cup of sugar” from another lab, either product you’re given will work just fine.

5. Ampicillin (Sodium) vs. Carbenicillin (Disodium)

    vs.     

Ampicillin is widely used for selection during cloning experiments; however it has its drawbacks. Carbenicillin is a more stable substitute, but like the previous examples, advantages always come with a cost. So do the benefits of carbenicillin outweigh that cost? And, are there times where one antibiotic is more advantageous than the other?

During selection, cells containing the bla gene from transformation will show resistance to ampicillin by expressing beta-lactamase, which inactivates ampicillin. The problem is that beta-lactamase is secreted by the bacterial cells, and when enough extracellular accumulation occurs, ampicillin in culture can be inactivated. What this ultimately means is that you can have a lower yield of desired cells in liquid culture, and satellite colonies may appear on agar plates (“These aren’t the cells you’re looking for”). As your plates age, the risk for satellite colonies increases.

Carbenicillin is more stable than ampicillin. It has a higher resistance to heat, it won’t degrade as easily in a lower pH, and it has an increased shelf life. But the other benefit is that satellite colonies are less likely to form since carbenicillin lasts longer and is less susceptible to hydrolysis by beta-lactamase. While all that sounds good, the risk when using carbenicillin is that its potency may kill cells before they have time to manufacture the resistance.

Verdict:
Keep both in the lab, and know when to use one over the other. When you’re dealing with quantification and longer incubation times, it’s safer to use carbenicillin. But when you’re doing a ligation, your cells are slow growing or your DNA is fragile, it’s safer to use ampicillin.

So there you have it, a starter guide for some common reagents that do very similar things. We know this is a list of only five categories. So if you think we need to have a part two, please chime in with your suggestions, questions or even your answers about other products!


    
              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: 88253 79108 79107

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

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

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

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

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

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

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

Triple Bioluminescence

 
 

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

Category Code: 79101 88231