Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments

Posted on by Ben Schmidt

In vitro translation of proteins through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function.  If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale).  There are many systems out there, and knowing which to use can sometimes be difficult.  Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA.  But when you want to make your own RNA templates to add to lysates, then there are additional concerns.

artists concept of in vitro translation
A protein chain being produced from a ribosome.

Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason.  Extracting it requires the utmost care in technique and elimination of nucleases.  Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease).  Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.

The first step for using cell-free expression systems with RNA templates is to make the RNA.  Here are some tips that will ensure success.

Continue reading “Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments”

Culture Rules- Investigating Company Cultures

Posted on by Becca McKnight

iStock_000025830858SmallWhen searching for a job it’s important to consider the job duties as well as the company and the company’s culture. Two companies have become famous for their cultures—Google and Zappos. Google is known as a company where you work hard in an amazing environment. Oh, and the food is free! Zappos is known as a place where employees are valued, and customer service is the first priority. Here at Promega, science rules, employee well-being is extremely important, and you can make a big impact regardless of your job title.

If you are able to find a company with an appealing culture and similar values to your own, it is a win-win situation. You will likely be happier in your job and therefore a better performer.

Here are some questions that you can ask to learn about the company culture and figure out if it is a fit with your personality and needs. These questions can be asked in an interview or in an informational conversation with someone in your network before you apply for a job. Keep in mind that there is no right answer to these questions. Some people thrive in government jobs while others have more of an entrepreneurial spirit; you need to figure out what type of culture will work best for you. Continue reading “Culture Rules- Investigating Company Cultures”

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.

Are you looking for proteases to use in your research?
Explore our portfolio of proteases today.

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”