Cytotoxicity Testing of 9,667 Tox21 Compounds using Two Real-Time Assays by Promega

A recent paper in PLOS One demonstrated real-time cytotoxicity profiling of approximately 10,000 chemical compounds in the Tox21 compound library, using two Promega assays, RealTime-Glo™ MT Cell Viability Assay and CellTox™ Green Cytotoxicity Assay. This is exciting to me, a science writer working at Promega; exciting because it’s tricky figuring out how to write about the utility of our products without sounding like an evangelist.

I don’t know about you, but I tend to shut out evangelists and their messages.

Instead of me telling you about real-time viability and cytotoxicity assays from Promega, here is an example of their use in Tox21 chemical compound library research.

What is the Tox21 compound library?
As described in the article by Hsieh, J-H. et al. (2017) in PLOS One:
“The Toxicology in the 21st Century (Tox21) program is a federal collaboration among the National Institutes of Health, including the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences and the National Center for Advancing Translational Sciences, the Environmental Protection Agency, and the Food and Drug Administration. Tox21 researchers utilize a screening method called high throughput screening (HTS) that uses automated methods to quickly and efficiently test chemicals for activity across a battery of assays that target cellular processes. These assays are useful for rapidly evaluating large numbers of chemicals to provide insight on potential human health effects.” Continue reading

Use of HIC high resolution chromatography and elastase for bottom up proteomics

One of the key applications used to characterize single or complex protein mixtures via bottom up proteomics is liquid chromatography−tandem mass spectrometry (LC−MS/MS).
Recent technical advances allow for identification of >10 000 proteins in a cancer cell line. On the peptide level chromatography methods, like strong cation exchange (SCX)
and hydrophilic interaction chromatography (HILIC), as well as high-pH reversed phase chromatography have been employed successfully. Because of its robustness
and ease of handling, the classical and still widely used approach for protein fractionation prior to LC− MS/MS is gel-based separation under denaturing conditions (SDS-PAGE).
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used
to study post-translational modifications of proteins and drug−protein interactions.  HIC is a high-resolution chromatography mode based on the interaction of
weakly hydrophobic ligands of the stationary phase with hydrophobic patches on the surface of the tertiary structure of proteins. By employment of high concentrations
of structure-promoting (“kosmotropic”) salts, proteins in HIC retain their conform

In a recent publication, HIC was used to separate proteins, followed by bottom up LC−MS/MS experiments (1).  HIC was used to fractionate antibody species
followed by comprehensive peptide mapping as well as to study protein complexes in human cells. The results indicated that HIC−reversed-phase chromatography (RPC)
mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

An additional observation noted that tryptic digests of the antibody used in the study yielded a protein coverage of 56% for the light chain and 63.2% for the
heavy chain. A consecutive proteolytic digestion protocol combing on-filter trypsin and elastase digestion drastically improved sequence coverage of
both light (100%) and heavy chains (99.2%).

Reference
1. Rackiewicz, M. et al. (2017) Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes. J.Proteome.Res. 16, 2318–23.

Promega Tech Tours 2017: The Power to Solve for the Forensic Community

Governor John Bel Edwards of Louisiana made an appearance at the Promega Technology Tour in Baton Rouge. Pictures courtesy of Forensic Scientists at the Louisiana State Police.

2017 finds Promega on the road visiting cities all across the United States. This year we are presenting workshops from leaders in the forensics community on topics like maximizing success with challenging samples, improving laboratory efficiency and reducing backlogs, and new tools and technologies for the forensics laboratory. This highly popular workshop series is a great way to learn from your peers about new techniques and workflows and network with other forensics experts in your region.

There are several more tours left between now and the end of 2017. Find out if we are coming to a city near you and register today!

Just in Time for Wisconsin’s Invasive Species Month: Goats

Invasive kudzu vine

Invasive kudzu vine covering a forest

“In Georgia, the legend says
That you must close your windows
At night to keep it out of the house
The glass is tinged with green, even so
As tendrils crawl over the fields…”
—James Dickey (1)

I grew up in Georgia, where on a hot, humid summer day you could almost hear the hiss of growing vegetation, especially the Kudzu as it climbed over fence posts and encroached upon the roadside, the king of invasive species. In Florida you worry about the alligators along the roadside if you have a flat tire; in Georgia, beware the Kudzu.

Invasive species, animal and plant, are an issue in all ecosytems. Imported from distant (and not-so-distant) areas both by accident and misguided intent, invasive species are species that have escaped the checks and balances of natural competitors and predators that existed in their native habitats. This lack of predation and competition enables them to outcompete and overrun other species.

Kudzu may be one of the most recognized invasive species in the United States, but it’s probably not the worst. The zebra mussel is an aquatic animal that has invaded our waterways in Wisconsin. Oak savannahs and prairie ecosystems in the Midwest United States are threatened by many invasive plant species like garlic mustard and blister parsnip. The Wisconsin DNR lists nearly 150 restricted and/or prohibited invasive plant species in Wisconsin, including Kudzu (2). Continue reading

Real-Time Analysis for Cell Viability, Cytotoxicity and Apoptosis: What Would You Do with More Data from One Sample?

You are studying the effects of a compound(s) on your cells. You want to know how the compound affects cell health over a period of hours, or even days. Real-time assays allow you to monitor cell viability, cytotoxicity and apoptosis continuously, to detect changes over time.

Why use a real-time assay?
A real-time assay enables you to repeatedly measure specific events or conditions over time from the same sample or plate well. Repeated measurement is possible because the cells are not harmed by real-time assay reagents. Real-time assays allow you to collect data without lysing the cells.

Advantages of  Real-Time Measurement
Real-time assays allow you to: Continue reading

All Aglow in the Ocean Deep

 

Fascinating bioluminescent creature floating on dark waters of the ocean. Polychaete tomopteris.

Today’s blog comes to you from the Promega North America Branch Office.

In nature, the ability to “glow” is actually quite common. Bioluminescence, the chemical reaction involving the molecule luciferin, is a useful adaptation for many lifeforms. Fireflies, mushrooms and creatures of the ocean deep use their internal lightshows to cope with a variety of situations. Used for hunting, communicating, ridding cells of oxygen, and simply surviving in the darkness of the ocean depths, bioluminescence is one of nature’s more flashy, and advantageous traits.

In new research published in April in the journal Scientific Reports, MBARI researchers Séverine Martini and Steve Haddock found that three-quarters of all sea animals make their own light.  The study reviewed 17 years of video from Monterey Bay, Calif in oceans that descended to 2.5 miles, to determine the commonality of bioluminescence in the deep waters.

Martini and Haddock’s observations concluded that 76 percent off all observed animals produced some light, including 97 to 99.7 cnidarians (jellyfish), half of fish, and most polychaetes (worms), cephalopods (squid), and crustaceans (shrimp).

Most of us are familiar with the fabled anglerfish, the menacing deep-sea creature known for attracting ignorant prey with a glowing lure attached to their head. As you descend below 200 meters, where light no longer penetrates, you will be surprised at the unexpected color display of the oceans’ sea life. Bioluminescence is not simply an exotic phenomenon, but an important ecological trait that the oceans’ sea creatures have wholeheartedly adopted to cope with complete darkness. Continue reading

Why wait ? Sample prep/protein digestion in as little as 30 minutes!

While many proteases are used in bottom-up mass spectrometric (MS) analysis, trypsin (4,5) is the de facto protease of choice for most applications. There are several reasons for this: Trypsin is highly efficient, active and specific. Tryptic peptides produced after proteolysis are ideally suited, in terms of both size (350–1,600 Daltons) and charge (+2 to +4), for MS analysis. One significant drawback to trypsin digestion is the long sample preparation times, which typically range from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove these compounds prior to analysis.

To shorten the time required to prepare samples for LC-MS/MS analysis, we have developed a specialized trypsin preparation that supports rapid and efficient digestion at temperatures as high as 70°C. There are several benefits to this approach. First, proteolytic reaction times are dramatically shortened. Second, because no chemical denaturants have been added, off -line sample cleanup is not necessary, leading to shorter preparation times and diminished sample losses.

The Rapid Digestion trypsin protocols are highly flexible. They can accommodate a variety of additives including reducing and alkylating agents. There are no restrictions on sample volume or substrate concentrations with these kits. Furthermore, the protocol is simple to follow and requires no laboratory equipment beyond a heat block. Digestion is achieved completely using an in-solution approach, and since the enzyme is not immobilized on beads, the protocol does not have strict requirements for rapid shaking and off -line filtering to remove beads.

In addition to the benefits of this flexibility, we also developed a Rapid Digestion–Trypsin/Lys-C mixture. Like the Trypsin/Lys-C Mix previously developed to prepare maximally efficiently proteolytic digests, particularly for complex mixtures, Rapid Digestion–Trypsin/Lys C is ideally suited for studies that require improved reproducibility across samples.

 

Surfing the Light Waves: Shrimp, Coral, Turtles and Other Fluorescent Organisms

A branching torch coral, Euphyllia glabrescens.

Have you ever walked on a beach and noticed that the waves seem to glow as they roll onto shore? Perhaps you have seen fish or jellyfish that glow in the dark, or maybe you’ve chased fireflies in your backyard or on a camping trip. These are all forms of luminescence (the production of light without adding heat), but the manner that these organisms produce their light can be quite different. Continue reading

In Healthy Eating Less is More: The Science Behind Intermittent Fasting

Mix a love of eating with a desire to live a long, healthy life what do you get? Probably the average 21st century person looking for a way to continue enjoying food despite insufficient exercise and/or an age-related decline in caloric needs.

Enter intermittent fasting, a topic that has found it’s way into most news sources, from National Institutes of Health (NIH) and Proceedings of the National Academy of Sciences publications to WebMD and even the popular press. For instance, National Public Radio’s “The Salt” writers have tried and written about their experiences with dietary restriction.

While fasting has enjoyed fad-like popularity the past several years, it is not new. Fasting, whether purposely not eating or eating a restricted diet, has been practiced for 1,000s of years. What is new is research studies from which we are learning the physiologic effects of fasting and other forms of decreased nutrient intake.

You may have heard the claims that fasting makes people smarter, more focused and thinner? Researchers today are using cell and animal models, and even human subjects, to measure biochemical responses at the cellular level to restricted nutrient intake and meal timing, in part to prove/disprove such claims (1,2). Continue reading

Biotech Manufacturing: A Good Machinist is Critical for Your Laboratory Reagents

Travis Beyer, Machinist Technician, at the CNC milling machine in the Promega machine shop.

Travis Beyer, Machinist Technician, at the CNC milling machine in the Promega machine shop.

It can be easy to forget that Promega is a manufacturing business. The company’s cGMP Feynman Center on the Madison campus has been described as looking more like a retreat than a factory. But hidden within the well-designed walls of Feynman, as well as in other facilities on campus, technicians operate hundreds of machines that manufacture, dispense and package Promega reagents day in and day out. Keeping those high-tech machines running at peak performance is critical, requiring immense skill, precision and even artistry. That’s where Promega Machinist Technician Travis Beyer comes in.

“I get to make stuff,” says Travis who is not afraid to show his enthusiasm for his craft while describing the best part of his job. “There’s a product at the end of the day. Plus I get to support science, and make things that support people’s lives. That’s cool.”

 I get to make stuff. There’s a product at the end of the day. Plus I get to support science, and make things that support people’s lives. That’s cool.

The da Vinci Center, another artfully designed building on the Madison campus, houses the Promega machine shop where Travis does his work designing or improving on parts for newer manufacturing equipment or reverse engineering broken or worn parts no longer available for older equipment that still serves its purpose. He makes every machine part imaginable from drive shafts to sensor brackets to filling forks, and his work is critical to manufacturing businesses like Promega, where a downed piece of equipment can cause costly production delays.

An example of a machine part that Travis designs or reverse engineers and then builds to keep Promega manufacturing moving smoothly.

An example of a machine part that Travis designs or reverse engineers and then builds to keep Promega manufacturing moving smoothly.

As he explains, not many manufacturing companies the size of Promega have a fully capable machine shop. They usually send out their work, meaning longer lead times and more expense. But, as its distinctive architecture suggests, Promega is not like many other companies. Continue reading