To inject or not inject?

Posted on by Ben Schmidt
GloMax® Discover Multimode Reader with injectors.
GloMax® Discover Multimode Reader with injectors.

Luciferase assays are useful tools for studying a wide range of biological questions. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. However, once this reagent has been added, how long you wait to measure the signal becomes a key consideration in generating consistent data. Dependent on which luciferase assay you use, you may need a luminometer that can use injectors to deliver the assay reagents. The reason for this is simple, but can be confusing to new users.

Let’s start by discussing two types of luciferase assays: “flash” vs. “glow”. Continue reading “To inject or not inject?”

DNA Reveals the Identity of Jack the Ripper?

Posted on by Terri Sundquist

A wanted poster for Jack the Ripper, who was also known as Leather Apron. Image courtesy of the British Museum
A wanted poster for Jack the Ripper, who was also known as Leather Apron.
Image courtesy of the British Museum

In the late 1800s, Victorian England was mesmerized and horrified by a series of brutal killings in the crowded and impoverished Whitechapel district. The serial killer, who became known as “Jack the Ripper”, had murdered and mutilated at least five women, many of whom worked as prostitutes in the slums around London. None of these murders were ever solved, and Jack the Ripper was never identified, although investigators interviewed more than 2,000 people and named more than 100 suspects. Now, 126 years after the murders, a British author, who coincidentally has just published a book on the subject, is claiming that DNA analysis has revealed the identity of the notorious killer. DNA is often thought to be the “gold standard” of human identification techniques, so why is there so much skepticism surrounding this identification?
Continue reading “DNA Reveals the Identity of Jack the Ripper?”

Fold It Up and Discover a Whole New World

Posted on by Michele Arduengo, PhD
FIGURE 1: Foldscope design, components and usage. (A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.
FIGURE 1: Foldscope design, components and usage.
(A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.

Scientific inquiry —looking at the world and asking questions about what we observe—is a natural human behavior. Why is the sky blue? What would happen if I did this Mom? Ask any grade school teacher. Kids do science naturally. They are not afraid of questions. They are not afraid of nature. They are not afraid of experiments and data collection.

One other things kids do really well is: fold paper. I never cease to be amazed at the elaborate fortune tellers, hoppers, boats, hats and other creations that my daughter and her friends make at a moment’s notice out of virtually any scrap of paper they can find.

Recently members of the Prakash Lab at Standford University announced the Foldscope: an optical microscope that is printed and folded from a single flat sheet of paper. These microscopes, which can provide magnification of up to 2000X, can be produced for less than $1.00/each. Furthermore these scopes weigh less than 10g (a couple of coins), require no external power source, can be dropped from 3-stories without damage, and can even be stepped on.

These characteristics make the Foldscope ideal for field work, particularly in remote locations where access to power and other resources is difficult. Prakash and colleagues have published their work in a PLOS One paper and have demonstrated many uses for these Foldscopes including high-resolution brightfield microscopy, fluorescence microscopy, and darkfield microscopy. Continue reading “Fold It Up and Discover a Whole New World”

Friday Fun on the Blog

Posted on by Promega

As we start a new year in our labs, we thought this cartoon reminding us of some lab etiquette might be useful. Enjoy!

 

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Promising Treatment for Marburg Virus Hemorrhagic Fever

Posted on by Sara Klink

I admit to some trepidation about the diseases that may be harbored in my backyard. For example, do the mice in my yard and, despite my and my cats’ efforts, in my house carry deer ticks that harbor the bacterium Borrelia burgdorferi, which causes Lyme disease? Should I be keeping an eye on the vitality of the birds around my property and density of my local mosquito population for potential risk of West Nile Virus transmission? As troublesome as these infections can be, mortality is low for infected humans. Contrast that with the mortality rate of up to 90% for the filoviruses Ebola and Marburg. I find it easy to dismiss these viruses because the reservoir (asymptomatic host) is not in the Upper Midwest but rather Africa, but the tragedy of the Ebola outbreak in the West African countries of Liberia, Sierra Leone and Guinea demonstrates the number of lives lost in an epidemic. Currently, there is no therapy or vaccine to treat these deadly viruses other than transferring antibodies from survivors to those infected. Therefore, the article in Science Translational Medicine about an antiviral treatment that protected macaques injected with a lethal dose of Marburg virus was welcome news.

Continue reading “Promising Treatment for Marburg Virus Hemorrhagic Fever”

Chikungunya Virus and the Promise of a Virus-Like Particle Vaccine

Posted on by Kelly Grooms

My family and I just returned from a week-long camping trip along the North Shore of Lake Superior in Minnesota. It is beautiful country, filled with lakes, rivers, ponds—and mosquitoes, lots and lots of mosquitoes. We went prepared for the worse. We had a screen tent, head nets and tubes and tubes of insect repellent because in this area of the world, mosquitoes are a flying, buzzing, picnic-ruining, itch-inducing pest. In the US, though, a pest is really all they are. In other areas of the world they are a flying, buzzing, disease-carrying, deadly menace.

Image courtesy of James Gathany and the CDC
Image courtesy of James Gathany and the CDC

Mosquitos act as vectors for many diseases including malaria, Dengue fever, Yellow fever, encephalitis, West Nile Virus and chikungunya virus. Many of these diseases are deadly; in fact, mosquitoes are responsible for more human deaths than any other animal (~725,000 deaths annually). Although most of these diseases have a long and infamous history, two of them, West Nile virus (first identified in 1932) and chikungunya virus (first identified in 1950), are relative new comers on the world health stage. Continue reading “Chikungunya Virus and the Promise of a Virus-Like Particle Vaccine”

Because Timing Is Everything….

Posted on by Robert Deyes

Camp Indian Trails Morning Flag CeremonyTiming is everything! I learned that the hard way just two weeks ago when I took my son to scout camp and thought I would try to capture the traditional American flag ceremony for posterity. I set up my camera for a panoramic shot and scanned the crowd.  Feeling very pleased with myself, I got home that evening ready to show my family the great camera skills I had honed over the Summer months. To my horror, I noticed that half of the scout troop was saluting the flag while the other half were standing to attention! I had got the timing horribly wrong (although the picture is still fun to look at in a strange sort of way).

Timing is everything in science as well.  As a technical services scientist at Promega I have sung the ‘timing’ tune to many a biologist. No more so than in the study of apoptosis where Caspases activate each other in a choreographed cascade of molecular triggers that all have their place and time in a domino sequence of enzymatic cleavage events. I frequently talk to researchers about that ‘sweet spot’ of activity when any given Caspase is busily cleaving a peptide moiety off of the next Caspase in the sequence. Finding that sweet spot is anything but trivial and often requires a considerable amount of patience during the optimization phase of experimentation.

Promega has developed a comprehensive suite of systems (see here) designed to help get the timing right for the cell and compound combinations you might be working with.  The end result is that you have experiments that are timed so as to give you reliable information about what is really happening in your cells.

Novel Cell Surface Markers Identified that Differentiate White, Beige and Brown Adipocytes

Posted on by Kari Kenefick

2012 CDC-based data on U.S. obesity percentages by state.
2012 CDC-based data on U.S. obesity percentages by state.

Estimates of obesity in the U.S. range from 30% (Centers for Disease Control data) to 70% (persons selling online and television audience-focused weight-loss programs). We are a nation of fat or fat-obsessed persons, and rightfully so. CDC data shows that the cost of obesity, in 2008 dollars, was estimated at $147 billion. That amount of money would buy a lot of french fries or cheesecake or __ (name your poison).

We all help pay those high-dollar amounts in terms of rising healthcare costs, thus there is considerable interest in finding ways to not only avoid, but also to combat obesity.

In recent years researchers working to understand body fat biology have produced exciting information on differences in types of fat. For instance, we now understand that in addition to white adipose tissue, animals and humans also have brown and beige adipose tissue. White adipose tissue or WAT is commonly found in humans and mice subcutaneously and in visceral fat. Brown adipose tissue or BAT, and beige adipose, is less common, and in humans and mice, is found in deeper cervical, supraclavical and paraspinal areas.

Continue reading “Novel Cell Surface Markers Identified that Differentiate White, Beige and Brown Adipocytes”

Use of Mass Spectrometry to Quantitate Food Allergens

Posted on by Gary Kobs

 

Food allergies are becoming increasingly prevalent among children. Credit: James Gathany, CDC
Food allergies are becoming increasingly prevalent among children. Credit: James Gathany, CDC

Food allergies are increasing worldwide and becoming a public health issue, especially among children are concerned. Children have a higher prevalence of food allergies, with about 4–8%, compared to adults (1–5%).  Currently antibody-based methods such ELISA (enzyme-linked immunosorbent assay) are the primary method for food allergen analysis. In most cases antibodies are only available for single well-known allergens. Often those that are commercially available are poorly characterized resulting cross-reactivity that leads to false-positive results in diagnostic tests.

A recent publication (1) presented a review of an alternative technology based on mass spec (i.e., multiple reaction monitoring, MRM) that circumvents the drawbacks of antibody based methods. MRM allows precise quantitative determination of target proteins in complex samples with broad dynamic range.  MRM also provides quantification of different isoforms. It is noted that tryptic digestion followed by mass spec analysis, has already identified several unique peptides for different allergens, including those found in crustaceans, eggs, fish, peanuts, soy and wheat. In summary the challenge is now to select the appropriate tryptic signature peptide(s) for the respective allergen and to develop well characterized standards (i.e., isotope labeled standards) to ensure accurate quanititation.

Citation:

Koeberl, M et al. (2014) Next Generation of Food Allergen Quantification using Mass Spectrometric Systems J. Proteome Research  13, 3499–509.

Freedom to Focus: Maxwell® Rapid Sample Concentrator

Posted on by Michele Arduengo, PhD
Wish I had one of these when I was at the lab bench...
Wish I had one of these when I was at the lab bench…

Back in the dark ages, when I was a graduate student, my idea of “automated” plasmid DNA extraction involved performing home-brew, “toothpick preps” in “strip tubes” or , if I was really feeling ambitious, a 96-well plate.

I would get just enough DNA to check for the presence of an insert, but the quality of the DNA was too low and the quantity too small to even consider using it for any other downstream experiments like amplification.

And increased throughput for other nucleic acid extraction needs? Nope. If I wanted genomic DNA, RNA or high-quality plasmid DNA, I spent time with columns and tubes, giving each sample my undivided individual attention.

Remember cesium chloride preps for RNA isolation? Even with the advent of column purification, which greatly simplified and standardized my protocols, nucleic acid purification was still a manual task that required a lot of time and effort to get the high-quality product I needed.

Doing the experiments that would answer the questions that I really wanted to ask (those “downstream experiments”), meant spending time at the bench performing careful (if tedious) work to isolate and clean up the highest quality nucleic acid possible. Even then inconsistency in sample prep could wreak havoc on downstream work.

Fortunately, for the modern scientist, personal, bench top automation, has progressed far beyond the toothpick and the strip tube to quality-tested, reliable nucleic acid extraction platforms like the Maxwell® Rapid Sample Concentrator (RSC).

The Maxwell® RSC improves sample preparation consistency, eliminating variability associated with manual handling, and your downstream results will reflect this consistency.  With the RSC you can extract DNA or RNA from up to 16 samples in approximately 1 hour and viral total nucleic acids in less than an hour.

The instrument is easy to use: simply load the sample, push a button and walk away. Cross contamination is minimized and the instrument is supported by the Promega technical support and service you have come to trust over the past 35 years. 

Want to know more about how the Maxwell® RSC can give you the freedom to focus on the work that interests you the most? To learn more, click here.