Asp-N Protease: Applications Update

Posted on by Gary Kobs
TrypsinLysC Page 2

Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for  in-solution or in-gel digestion reactions.

The following references illustrate the use of Asp-N in recent publications:

Protein sequence coverage

  1. Jakobsson, M et al. (2013)  Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
  2. Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I.  J. Biol. Chem. 288, 24799–08.

Glycoprotein analysis

  1. Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
  2. Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
  3. Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”.  J. Biol. Chem. 288, 30192–09.

Phosphoprotein analysis

  1. Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.

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Get More Out of Your Lentiviral Production

Posted on by Promega

fugene6_lvv_blogThis review is a guest blog by Amy Landreman, Product Specialist in Cellular Analysis at Promega Corporation.

Lentiviral vectors (LVV) have become a valuable research tool for delivering genetic content into a wide range of cell types. Commonly derived from the HIV-1 genome, LVV have the advantage of being able to infect both dividing and non-dividing cells. They can be particularly valuable for introducing genetic material into cell lines that are difficult to transfect using other methods and are also being used in gene therapy applications.

Unlike other gene delivery tools, transducing mammalian cells with LVV requires significant upfront effort since the LVV particles carrying the desired genetic content first need to be created. In general this involves co-transfecting a packaging cell line, such as HEK293T, with a set of three to four separate plasmids that encode the protein content required to generate the LVV particles: the transfer plasmid, which contains the transgene of interest, a packaging plasmid, and an envelope plasmid. After co-transfection, the packaging cell line is allowed to incubate for a couple of days during which time the LVV particles are produced and accumulating in the culture supernatant. The supernatant containing the recombinant LVV is then harvested and, following several concentration steps, the LVV particles are ready to be used for introducing the desired genetic content into the mammalian target cells. Continue reading “Get More Out of Your Lentiviral Production”

Not Music to Everyone’s Ears

Posted on by Anupama Gopalakrishnan

iStock_000016543302SmallWhen my son was about 2 years old, he commented that the jingles “Twinkle twinkle little star” and “alphabet song” had the same musical notation. While I do not think I am tone deaf and I do appreciate music, I had not made the connection in all these years.  Music appreciation is perhaps one of the most subjective and controversial topics. For some people, appreciating music involves understanding the technical nuances and critically evaluating artist’s mastery over the art, and for some of us, it is about simply enjoying the patterns and rhythms. While one might claim that they enjoy all kinds of music, for most of us, only certain kinds of music elicit a deeper appreciation, emotive experience and pleasure. Our music preferences are molded by exposure, cultural diversities and to some extent, mood. Music is extremely varied, and listing the kinds of music could fill pages. Arguing one kind of music is better than other is as like saying one color is better than the other.

So, what biological purpose does music serve? Continue reading “Not Music to Everyone’s Ears”

Ghosts

Posted on by Sam Jackson

buddhabrot fractal image
Formless, recursive and abstract, it can be tough to wrestle the ghosts of the conscious mind.

Please believe me when I say this is the hardest thing I’ve done. Typing this sentence might as well be lifting a boulder, and the next could be even heavier. Before this, the hardest thing I’d done was say “good morning” to co-workers, and before that, it was simply getting out of bed.

Just about the only thing I find easy is going to bed, but sleeping is a different story. Every night I lie down, unsure if I’ll fall asleep within seconds and wake what seems like moments later, swatting aimlessly at my alarm clock, or if I’ll remain awake, tired beyond belief but some mysterious finger in the dyke preventing a flood of sleep from washing over me.

I’m one of the approximately 21 million people in the United States who suffer from major depression. Let me tell you, it’s kind of a bummer. Lying awake at night might sound terrible, but it’s the easiest thing in the world compared to writing a sentence, saying “hello”, smiling. I live each day negotiating a watery fog, often unsure what people tell me, confused about what comes next, and desperate for the energy to participate in the world.

This isn’t an essay asking for sympathy; receiving pity from others would only make me feel worse. Besides, as a function of suffering from depression, I’m convinced nobody is reading this, that nobody is going to read this. This essay is for me. Only by engaging and grappling with this disease in words and in actions can I ever hope to pin it to the ground.

Continue reading “Ghosts”

Dark Chocolate Benefits Improved by Fiber

Posted on by Kari Kenefick

Add pomegranate to your chocolate, says researcher Finley, to aid it's digestion, health benefits.
Add pomegranate to your chocolate, says researcher Finley, to aid it’s digestion and health benefits.

For chocolate lovers (and chocolate makers) it has been a great decade or so. Scientific research continues to prove what our brains have been saying for years; chocolate really IS good for us.

Research over the past decade or so has studied dark chocolate and its polyphenolic compounds, such as catechin and epicatechin, for their effects on inflammation, and cardiac and endothelial cell function. Today, from the American Chemical Society meeting in Dallas, TX, we learn new details about how dark chocolate brings its health benefits.

Before beneficial compounds in dark chocolate can reach the heart and other tissues in the body, digestive processes must occur to release the beneficial compounds from the chocolate.

Researcher John Finley and cohorts from Louisiana State University created a model digestive system by which to study what happens when cocoa combines with typical gut bacteria.

Their research showed that bacterial species in the colon ferment the fiber found in cocoa, which in turn aids in digestion of the larger polyphenols in cocoa, into smaller, more easily absorbed molecules. These smaller molecules, the catechins and epicatechins then enter the bloodstream and exert their anti-inflammatory effects.

Finley emphasized the role of dietary fiber, such as the fiber in the cocoa powders tested in this research, in the digestion process. He noted that prebiotics, carbohydrates in foods like raw garlic or cooked whole wheat flour, while not digested by humans, aid digestion and absorption of healthful food components, in this case polyphenols in dark chocolate.  Continue reading “Dark Chocolate Benefits Improved by Fiber”

Uncovering the Life and Death of a Mummy

Posted on by Sara Klink
Frontal view of the mummy
Panel A of Figure 1 shows the frontal view of the mummy. Panzer, S. et al. (2014) Reconstructing the Life of an Unknown (ca. 500 Years-Old South American Inca) Mummy – Multidisciplinary Study of a Peruvian Inca Mummy Suggests Severe Chagas Disease and Ritual Homicide. PLoS ONE 9(2): e89528. doi:10.1371/journal.pone.0089528

Deciphering how an ancient person lived and died is based heavily on the context of the buried body or mummy, including the soil around the gravesite, artifacts present in the grave and if nothing else, the location of the remains. What happens when there is no context? With skeletal remains or even bone fragments, which is primarily what is found at many burial sites, there is some information that can be derived but mummies that include tissue as well as bone offer a greater opportunity to learn about a deceased individual’s life. A recently published PLOS ONE article of a bog body identified using analyses across several scientific fields demonstrates how we can uncover the story of a person who lived several centuries ago based on her mummified remains.

Continue reading “Uncovering the Life and Death of a Mummy”

How to Isolate RNA like a Pro

Posted on by Terri Sundquist

Ribbon diagram of RNA’s biggest threat: a ribonuclease
Ribbon diagram of RNA’s biggest threat: a ribonuclease

Back in graduate school, I purified a lot of RNA, and after a while, I became fairly successful at it. My yields were good, and the RNA was intact. However, many of my early attempts at RNA isolation yielded degraded RNA that did not work well in many downstream applications. In my case, successfully isolating high-quality RNA required practice. During my trials and tribulations, I learned a lot of tricks and tips about how to obtain high-quality RNA. Here I share some of these tricks to help you speed through that “practice makes perfect” phase so that you can isolate RNA like a pro.

Continue reading “How to Isolate RNA like a Pro”

Elegant Experiments that Changed the World

Posted on by Isobel Maciver

Crystallographic structure of HIV reversed transcriptase. Wikimedia Commons
Crystallographic structure of HIV reverse transcriptase. Wikimedia Commons

Today, reverse transcriptases are commonplace molecular biology tools, easy to obtain and routinely used in labs for everyday cloning and gene expression analysis experiments. Reverse transcriptase inhibitors have also found widespread use as antiviral drugs in the treatment of retroviral infections.

It’s easy to forget that the existence of reverse transcriptase activity—the ability to convert an RNA template into DNA—was once a revolutionary notion not easily accepted by the scientific community. The idea that RNA could be the template for DNA synthesis challenged the “DNA–>RNA–> Protein” central dogma of molecular biology.

The foundational studies that proved the existence of a reverse transcriptase activity in RNA tumor viruses were described in two papers published back-to-back in Nature in June, 1970. Two of the authors of these studies, Howard Temin of the University of Wisconsin and David Baltimore of the Massachusetts Institute of Technology, were awarded a Nobel Prize for their work in 1975.

In appreciation of the significance of these papers, the editorial introduction published in Nature at the time states:

This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted.

Before these papers were published, it was known that successful infection of cells by RNA tumor viruses required DNA synthesis. Formation of virions could be inhibited by Actinomycin D—an inhibitor of DNA-dependent RNA polymerase—so it was known that synthesis of viral RNA from a DNA template was part of the viral life cycle. The existence of an intracellular DNA viral genome was therefore indicated, and had been postulated by Temin in the mid 1960’s. However, proof of the mechanism whereby this DNA template was generated from the RNA genome of the infecting virus remained elusive. Continue reading “Elegant Experiments that Changed the World”

5 Signs You’re Ready to Earn that Ph.D.

Posted on by Karen Reece

confused5. No one in your family bothers to ask you what you’re studying anymore.

The longer you spend in a Ph.D. program the more opportunities your family has to ask you what you’re doing in school. No doubt, you’ve spent the first couple years of graduate school going to family functions and trying to explain to your grandma what a molecule is. She will eventually come up with an explanation of what you are doing that she can share with her friends. Her description of your work may or may not be correct, but she’s not going to bother trying to understand it anymore. “Good for your honey, you’re so smart!”

4.  Your former life as a bartender or grocery store clerk starts to sound really appealing. Continue reading “5 Signs You’re Ready to Earn that Ph.D.”

From Soot, Turpentine and Walnut Oil to Bio-Inks with Living Cells: Printing Reinvents Itself in 3D and Comes Alive

Posted on by Kelly Grooms

Gutenberg-Style Printing Press.
Gutenberg-Style Printing Press (Replica). Wikimedia Commons.

Printing has been an integral part of society since the first movable type printing press was invented by Johannes Gutenberg in the 15th century. And yet, many recent headlines have heralded its impending death.  As everything from books to newspapers transitioned to digital media, the traditional printing techniques seemed destined for the history books and museums. However, even before the popularity of printing started to fade, some of the concepts were being reimagined in amazing, new—and three dimensional— ways. Continue reading “From Soot, Turpentine and Walnut Oil to Bio-Inks with Living Cells: Printing Reinvents Itself in 3D and Comes Alive”