Broaden Your Horizons While Pursuing Your Doctorate—You Will Be Glad You Did

Posted on by Amy Prevost, PhD

For this posting, I had promised to include some commentary on ACTION.

image credit: ComiCONNMitch via Wikimedia Commons
image credit: ComiCONNMitch via Wikimedia Commons

What can someone pursuing a doctorate in the biosciences DO during that time to widen the possibilities of employment in the future? In general, the process of obtaining the doctorate has been criticized for taking too long and not doing enough to prepare students for what they will do when they graduate. Considering these criticisms, it seems wrong to create additional check-boxes on the student to-do list leading up to graduation. Therefore, these things are not in addition to what is already expected, but are instead the same things that are already happening re-focused. Continue reading “Broaden Your Horizons While Pursuing Your Doctorate—You Will Be Glad You Did”

She’s Going Soft! – A commentary on “hard” and “soft” sciences

Posted on by Karen Reece

scientific-methodThis week I gave notice that I would be terminating my employment at Promega. This was a very difficult decision as I have really enjoyed the past six years here.  While I am leaving Biotech, I will not be leaving science all together.  Over the past few years, I have used my research, analytical, and organizational skills to assist various non-profit organizations in the community.  My primary focus will be on reform of the criminal justice system and racial disparities.  Spreading the word about this decision has resulted in a number of responses (overwhelmingly positive) including the comment that I am going soft! This got me thinking about where the terms hard and soft science came from. Continue reading “She’s Going Soft! – A commentary on “hard” and “soft” sciences”

Fun Friday: Western Blot Cartoon

Posted on by Kelly Grooms

As a fun followup to Wednesday’s blog, here is a cartoon for all you protein scientists out there.

Western Blot Cartoon by Ed Himelblau.
Western Blot Cartoon by Ed Himelblau.

IF you would like to see more science cartoons by Ed Himelblau, you can find them in the Cartoon Lab section of the Promega PubHub.

Discovering the Complexity of the Human Proteome

Posted on by Kelly Grooms

Transcription TranslationI should preface this blog by stating that I am a nucleic acids gal. My years in the lab were spent with tubes of DNA and RNA. In fact my one and only tentative foray into the field of protein resulted in a Western Blot so ugly that those who witnessed it have been sworn to secrecy. Given all of this, the mapping of the human proteome might seem like an odd topic for me to write about. Except that it isn’t really, because the sequencing of the proteome offers answers to some of the questions that the sequencing of the genome didn’t.

First, let’s start with what a proteome is: A proteome is all the proteins expressed at a certain time point. It can be as limited as the proteome of a single cell or as all encompassing as the proteome of an entire genome. However, unlike the genome, which is genetic information encoded in an organism’s DNA or RNA, the makeup of a proteome can vary dramatically as a result of expression patterns, alternative splicing events and post-translational modifications.

The genome is a constant, what you see today is what will still be there tomorrow. The proteome, on the other hand, is a constantly changing landscape. Up regulation or down regulation of a gene can mean more or less protein is present. Alternative splicing and post-translational modifications can result in fundamental changes to the protein itself.

In other words, if the genome is a beautiful, pristine Ansel Adams print, then the proteome is that same scene as interpreted by Andy Warhol—in Technicolor and 3D. Continue reading “Discovering the Complexity of the Human Proteome”

PowerQuant System: Tool for informed casework sample processing decisions

Posted on by Anupama Gopalakrishnan

CottonSwabsIdentification of a crime perpetrator on the basis of DNA fingerprinting is not as easy as some of the CSI shows on television make it out to be. A sample such as blood stain, touch sample or body fluid retrieved at a crime scene is often a challenge for DNA analysts. In many instances, the samples are limited in quantity, found in dirty conditions, exposed to harsh environmental factors and are mixtures of more than one DNA—human and/or non-human. One of the most important aspects of the workflow to successfully obtain a DNA fingerprint profile is accurate quantification of human amplifiable DNA. The more information gleaned from the sample, the better equipped the DNA analyst is to determine the best course of action for obtaining a usable short tandem repeat (STR) profile from challenging samples. Therefore, Promega has developed the PowerQuant™ System, a probe-based 4-target, 5-dye real-time PCR method to a) determine human and male DNA concentrations in a sample, b) detect possible PCR inhibitors c) identify possible mixtures and d) measure DNA integrity. Continue reading “PowerQuant System: Tool for informed casework sample processing decisions”

Angel’s Glow: Bioluminescence Uncovered on the Battlefield

Posted on by Radhika Ganeshan

New information has surfaced about this story, and we encourage you to read our updated blog from July 2024 (linked) for the latest on this story.

1888 Chromolithograph of the Battle of Shiloh, American Civil War, produced by L. Prang & Co.
1888 Chromolithograph of the Battle of Shiloh, American Civil War, produced by L. Prang & Co.

If battlegrounds could speak they would have many stories to tell.  In some cases the microbes found in those soils have lived on to separate fact from fiction. One such story has its origins in the Battle of Shiloh, which went down in history as one of the bloodiest battles fought during the American Civil War.  As the soldiers lay mortally wounded on the cold, hard grounds of Shiloh waiting for medical aid, they noticed a very strange phenomenon. Some of the wounds actually appeared to be glowing in the dark casting a faint light into the darkness of the battlefield. And the legend goes that soldiers with the glowing wounds had a better chance at survival and recovery from infections than their fellow brothers-in-arms whose wounds were not similarly luminescent. The seemingly protective effect of the mysterious light earned it the moniker “Angel’s Glow.”

Fast forward to the 21st century.

Continue reading “Angel’s Glow: Bioluminescence Uncovered on the Battlefield”

Purifying HIS-Tagged Proteins from Insect and Mammalian Cells

Posted on by Gary Kobs

MSextractcroppedMany different polypeptide fusion partners or affinity tags have been developed to facilitate purification of target proteins. The most commonly used tag for the purification and detection of recombinant expressed proteins is the His tag. Cloning vectors designed to generate His-tagged proteins contain 5–10 histidine residues at either the C- or N terminus of the expressed protein. The His tag adds only 0.84kDa to the mass of the protein and is nonimmunogenic. Also, because the tertiary structure of the tag is not important for purification, His-tagged proteins can be purified using native or denaturing conditions. The affinity of histidine residues for immobilized nickel allows selective purification of His-tagged proteins. The MagneHis™ Ni-Particles can bind up to 1mg of His-tagged protein per milliliter of particles providing a fast, efficient method for purifying His-tagged proteins with high yield and low background in a highly scalable format.

Bacterial expression of recombinant His-tagged proteins is a common technique. However, use of other systems, such as Sf9 insect cells,or HeLa or CHO mammalian cells for expression of recombinant proteins either intracellularly or secreted into the culture medium is increasing. These eukaryotic expression systems may allow more natural processing and modification of recombinant His-tagged proteins.
The following article:  illustrates the use of FastBreak™ Cell Lysis Reagent and the MagneHis™ Protein Purification System with insect and mammalian cell lysates. Proteins are purified from culture medium in the presence or absence of serum with only minior modifications to the standard protocol for bacterial cultures are required for purification from these diverse sources.
4550MA-(1)

 

How a Magazine Ad Helped Convict a Rapist

Posted on by Terri Sundquist
Trial

In May of 1986, a woman in Orange County, Florida, was surprised by a man who entered her apartment and raped her at knifepoint. Despite the fact that she got a glimpse of his face, the chances of identifying and convicting her rapist were slim. Although law enforcement officers did their best to identify the perpetrator, their investigative techniques in the case were limited compared to our current set of forensic tools. That changed when Jeffrey Ashton, an assistant attorney for the state of Florida, saw an advertisement for DNA-based paternity testing in a magazine and began to wonder if DNA testing could also be used to identify the man responsible for the attack.

Continue reading “How a Magazine Ad Helped Convict a Rapist”

Monitoring Mass Spec Instrument Performance and Sample Preparation

Posted on by Michele Arduengo, PhD

Proteomics, the analysis of the entire protein content of a living system, has become a vital part of life science research, and mass spectrometry (MS) is the method for analyzing proteins.  MS analysis of protein content allows researchers to identify proteins, sequence them and determine the nature of post translational modifications.

LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.
LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.

Mass spectrometry allows characterization of molecules by converting them to ions so that they can be manipulated in electrical and magnetic fields. Basically a small sample (analyte) is ionized, usually to cations by loss of an electron. After ionization, the charged particles (ions) are separated by mass and charge;  the separated particles are measured and data displayed as a mass spectrum. The mass spectrum is typically presented as a bar graph where each peak represents a single charged particle having a specific mass-to-charge (m/z) ratio. The height of the peak represents the relative abundance of the particle. The number and relative abundance of the ions reveal how different parts of the molecule relate to each other.

For the study of large, organic macromolecules, matrix associated laser desorption/ionization (MALDI) or tandem mass spec/collision induced dissociation (MS/MS) techniques are often used to generate the charged particles from the analyte. MS analysis brings sensitivity and specificity to proteome analysis. The technique has excellent resolution and is able to distinguish one ion from another, even when their m/z ratios are similar. Macromolecules are present in extremely different concentrations in the cells, and MS analysis can detect biomolecules across five logs of concentration.

Continue reading “Monitoring Mass Spec Instrument Performance and Sample Preparation”

Hope for Treatment of Carbapenem-Resistant Bacteria

Posted on by Isobel Maciver

Structure of the antibiotic meropenem
Structure of the antibiotic meropenem
Last month brought some hopeful news on the subject of antibiotic resistance. A paper published in Nature on June 26 described the isolation of a fungal compound that restored the antibiotic sensitivity of carbapenem-resistant enterobacteria. An editorial accompanying the paper took encouragement from the article–considering it a sign that the well of potential sources of new antimicrobial agents, and agents that inhibit resistance mechanisms, is not yet dry:

But the reservoir of natural products with the potential to act as antibacterial drugs has not yet been exhausted. In contrast to general thinking by drug companies, screening for such products may well still have a bright future” Nature News and Views: “Antibiotic resistance: To the rescue of old drugs” Meziane-Cherif & Courvalin, Nature 510, 477–478.

The emergence of bacteria that are resistant to antibiotics has been an object lesson in the relentlessness of natural selection; the moment a new antibiotic is developed and introduced, the countdown to the emergence of resistance begins. The race to keep the one step ahead of emerging resistance mechanisms has been going on since antibiotics were first introduced.

The history of the development of penicillin and related antibiotics is both an illustration of the ingenuity of scientists and of the never-ending nature of this battle with emerging resistance. The Nature paper is the latest installment in that story. Continue reading “Hope for Treatment of Carbapenem-Resistant Bacteria”