Making It Easier to Investigate PGTs

Posted on by Sara Klink

Introduction

Studying cellular molecules can be challenging. Some processes are troublesome to study due to the lack of an assay or a complicated assay exists but lacks sensitivity. Membrane proteins in particular are difficult to isolate and characterize. Phosphoglycosyltransferases (PGTs) are transmembrane proteins that transfer phosphosugars onto phospholipids, initiating the synthesis of oligosaccharides in bacterial cell walls. This transfer creates a diphosphate link between a lipid and a sugar and generates UMP as a byproduct. Once this lipid–P–P–sugar linkage occurs, more sugars can be added by glycosyltransferases, generating membrane-based polysaccharides (e.g., peptidoglycan) used for signaling, recognition and defense.

While PGTs have been studied biochemically and an X-ray structure of one member exists, much is still unknown about these enzymes. Overexpressing and purifying membrane proteins remains a challenge, and the conventional PGT assay requires isotope labeled-UDP-sugar donors and is based on the solubility difference between substrate and product to determine enzyme turnover using extraction-based or chromatographic methods. While there are other assays that use fluorescent modified substrates or multienzyme analysis, none of the methods can be applied to all of the diverse PGT enzymes.

All PGTs generate UMP as a byproduct of the transfer of a phosphosugar to a phospholipid. Based on the principle of the luminescent UDP-Glo™ Glycosyltransferase Assay where UDP released during the glycosyltransferase reaction was quantitated, a new luminescent assay called UMP-Glo™ Assay is able to measure the activity of PGT enzymes by adding a single reagent to detect UMP. Das et al. validated this assay by testing PglC, a PGT from Campylobacter jejuni, as well as PglC from Helicobacter pullorum and WecA from Thermatoga maritime and published the results in Scientific Reports. Continue reading “Making It Easier to Investigate PGTs”

Research Teams Demonstrate Bivalent Binding of a Novel Bromodomain Protein Inhibitor

Posted on by Promega
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Today’s blog is written by guest blogger Kristin Huwiler from our Cellular Analysis and Proteomics Group.

Two research collaborations, one in Europe and a second in the US, have just published in Nature Chemical Biology (1,2) on the identification of BET inhibitors (bi-BETs) that bind via a bivalent mechanism to both bromodomains of BRD4. These bivalent chemical inhibitors exhibit high cellular potency and affinity relative to their monovalent predecessors. By developing high-affinity ligands that engage both bromodomains simultaneously within BRD4, the authors illustrate a concept that may be applicable in the development of selective, potent ligands for other multi-domain proteins. Here we review the work presented in the Waring et al. paper using the Promega NanoBRET™ Technologies to characterize the mechanism of action of their bivalent probe.

The bromodomain and extraterminal (BET) sub-family are some of the most studied bromodomain-containing proteins (3). The BET subfamily of proteins contain two separate bromodomains. BRD4 is one well studied member of the BET sub-family. Several small molecule inhibitors that target BRD4 have been developed as potential therapeutics for various cancers with promising initial studies, but to date are all monovalent, binding each bromodomain of the BET family members separately (2).

Continue reading “Research Teams Demonstrate Bivalent Binding of a Novel Bromodomain Protein Inhibitor”

Improved Method for the Rapid Analysis of Monoclonal Antibodies Using IdeS

Posted on by Gary Kobs
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Therapeutic monoclonal antibodies (MAbs) are inherently heterogeneous due to a wide range of both enzymatic and chemical modifications, such as oxidation, deamidation and glycosylation which may occur during expression, purification or storage. For identification and functional evaluation of these modifications, stability studies
are typically performed by employing stress conditions such as exposure to chemical oxidizers, elevated pH and temperature.

To characterize MAbs, a variety of analytical techniques are chosen, such as size exclusion chromatography and ion exchange chromatography. However, due to the large size of the intact MAbs, these methods lack structural resolution. Often, the chromatographic peaks resolved by SEC and IEC methods are collected and further analyzed by peptide mapping to obtain more detailed information. Peptide mapping, in which antibodies are cleaved into small peptides through protease digestion followed by LC–MS/MS analysis, is generally the method of choice for detection and quantitation of site-specific modifications. However sample preparation and lengthy chromatographic separation make peptide mapping impractical for the analysis of large numbers of samples. In contrast to peptide mapping analysis, the middle-down approach offers the advantage of high-throughput and specificity for antibody characterization.

Limited proteolysis of IgG molecules by the IdeS enzyme has been introduced for antibody characterization due to its high cleavage specificity and simple digestion procedure.

Continue reading “Improved Method for the Rapid Analysis of Monoclonal Antibodies Using IdeS”

How’s That for Wellness?

Posted on by Molly Lenzendorf

Copyright: pavel1964 / 123RF Stock Photo
Copyright: pavel1964 / 123RF Stock Photo

Whether you’re a seasoned runner or just trying to find a fun activity to do with a coworker, a 5K run/walk can be a fun event for all.  Promega recently hosted its 7th annual 5K run/walk and had more than 155 participants at the Madison, WI,  location.  The event was a fun activity for everyone to get out, get active and be social. The event also helped raise over 160 pounds of food and over $115 in cash that was donated to the Second Harvest Food Bank.

As a nation, we have become more focused on our health and wellness than ever before. Nearly everyone is trying to drink more water, be more mindful of what they eat, and it’s hard to look around the room without finding someone wearing a fitness tracker to keep track of their daily steps.  Health and wellness has also become increasingly popular in the workplace. According to Health Fitness Revolution, the top benefits of a Workplace Wellness Program are as follow:

  • Fun
  • Improved Productivity
  • Happiness
  • Sense of Community
  • Lower Healthcare Costs
  • Sense of Accomplishment
  • Improved Physical Fitness
  • Weight Loss
  • Less Stress
  • Healthier Habits

At Promega our focus on wellness as an organization is no different. We recognize that being active and taking care of your health, and your family’s health, has an impact beyond how you feel.  Because of that, we have a wellness or fitness center in each of our facilities to make staying active and fit more convenient. We also believe that wellbeing extends beyond physical health and have a campus that features native gardens with walking paths and dedicated meditative spaces to encourage total wellness. Promega strives to be a leader in health & wellness initiatives that enable our employees to become the best version of themselves. We don’t do this for a potential monetary return on the investment, but because we truly believe that by becoming the best version of yourself, our employees also become the most fulfilled.

 

Back to Basics: Organizing Your Writing like It’s a Hamburger

Posted on by Michele Arduengo, PhD
The "hamburger" scheme for organizing a paragraph.
The “hamburger” scheme for organizing a paragraph.

Last night I was helping my daughter, who is in fourth grade, with her homework. We had completed a math worksheet, a geography worksheet and had moved onto writing. For her paragraph assignment, she was supposed to write about a special place. So I began drawing the concept map that we typically use to help her organize her thoughts. She stopped me before I could get started.

“No Mom, wait,” she grabbed the pencil and paper from my hands, “I have a better idea.”

She drew five shapes on the paper.

“We should write the paragraph like it’s a hamburger. The first sentence is the topic—it’s the top of the burger, tells you what is inside—it makes you hungry to read more. Next comes the juicy, meaty part. Three details—three sentences. Then the bottom bun, the summary that supports the whole paragraph. It’s the hardest to write.” She proudly sat down with her drawing and pencil.

“I LOVE that,” I exclaimed. “That’s a great way to organize a paragraph.”

“Yeah,” my husband looked up from his Suduko that he had been working on, “and the cheese goes right here.” He pointed to one of the three boxes my daughter had drawn underneath the bun.

“And the lettuce over here,” my daughter giggled.

“Well, I like mine with lettuce and tomato,” I chanted with no apologies to Jimmy Buffett, “Heinz 57 and French-fried potato..,”

“A big kosher pickle,” my daughter joined in, and the evening’s homework activities degenerated from there. (Sometimes it’s the parents who are easily distracted.)

My daughter’s hamburger graphic was new to me, but the concept wasn’t. It is a solid method for organizing a piece of writing, and it can be applied all kinds of writing—from a paragraph, to an essay, to a speech and even to a scientific article.

Continue reading “Back to Basics: Organizing Your Writing like It’s a Hamburger”

Of Elephant Research and Wildlife Crime – Molecular Tools that Matter

Posted on by Nicole Sandler

Here at Promega we receive some interesting requests…

Take the case of Virginia Riddle Pearson, elephant scientist. Three years ago we received an email from Pearson requesting a donation of GoTaq G2 Taq polymerase to take with her to Africa for her field work on elephant herpesvirus. Working out of her portable field lab (a tent) in South Africa and Botswana, she needed a polymerase she could count on to perform reliably after being transported for several days (on her lap) at room temperature. Through the joint effort of her regional sales representative in New Jersey/Pennsylvania (Pearson’s lab was based out of Princeton University at the time) and our Genomics product marketing team, she received the G2 Taq she needed to take to Africa. There she was able to conduct her experiments, leading to productive results and the opportunity to continue pursuing her work.

Continue reading “Of Elephant Research and Wildlife Crime – Molecular Tools that Matter”

Next-Generation Genomics Education: Educating and Preparing the Next Generation

Posted on by Amy Prevost, PhD

Students pursuing their interests wit h hands-on activities at BTC Institute.
Students pursuing their interests wit h hands-on activities at BTC Institute.

The BTC Institute has many partners in creating educational opportunities in the molecular biosciences. In recent years, we have worked with the Dane County School to Work Consortium (DCSWC) to create a unique, one-semester class aimed at giving high school Juniors and Seniors interested in scientific research and health careers a chance to explore how concepts they have been learning about in their biology and biotechnology classrooms are used in the laboratory.

To take it one step further, these laboratory experiences are tied to the Gates Foundation Grand Challenges in Global Health. The Challenges provide a framework to help students understand worldwide concerns, including ways in which biotechnology can be applied to generate solutions to these problems. Students are encouraged to place scientific challenges within social and socioeconomic contexts which, in turn, make some solutions more appealing than others. This holistic approach provides the “real world” milieu that is so sought after in academic endeavors. Continue reading “Next-Generation Genomics Education: Educating and Preparing the Next Generation”

Friday Cartoon Fun: Take My Samples, Please!

Posted on by Sara Klink

Instruments can make our lives easier in the lab. Place your samples inside an instrument and let it do all the work—isolating nucleic acids or reading and analyzing a multiwell plate—while you walk away to read a new research paper or prepare for the next step in your experiment. However, with the array of machines now available to scientists worldwide, some confusion may result in the laboratory. Has this ever happened to you?

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Copyright Ed Himelblau

The Birth of a Disease? A New Anthrax-Like Disease Found in Sub-Saharan Africa

Posted on by Kelly Grooms

Copyright
Copyright

It usually starts with one; one dead animal, one sick individual, one case that a doctor thinks is unusual. These are all ways that a new disease makes its presence known. In the case of Bacillus cereus biovar anthracis, it started with a dead chimpanzee (1).  

The wild western chimpanzee was found dead in Côte d’Ivoire in 2001. An investigation led by scientists from the Robert Koch Institute in Berlin identified the pathogenic cause of death to be an atypical B. cereus isolate that caused an anthrax-like disease. Continue reading “The Birth of a Disease? A New Anthrax-Like Disease Found in Sub-Saharan Africa”

Exploring the Land of the Silver Fern—Part II

Posted on by Promega

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part II of the adventures of Sarah Theos, a client support consultant, who used her award to travel to New Zealand. You can read Part I here.

Day 7: Queenstown to Te Anau

My husband surprised me that morning by booking a massage appointment at the spa to soothe my aching muscles. It was a rainy morning and we had a long drive ahead of us to the town of Te Anau, the stopover town on the way to the Fiordland National Park and Milford Sound.  We also saw that we had lucked out on our itinerary as it was supposed to be a beautiful day in Milford the next day (a place that gets over 300 days of rain each year).  Therefore, we didn’t mind one more day of rain if it meant the next day would be amazing.  We drove through the Canterbury plains, passing tons of sheep and dairy farms along the way.  We also found it interesting that they farm deer in NZ.  We drove past a lot of deer farms.  When we arrived in Te Anau, we grabbed some food and went to the local grocery store to stock up on snacks and more wine.  New Zealand has a booming wine industry and we thought that it was worth sampling as many wines as we could.  The day was gray and cold but we decided to walk along Lake Te Anau anyway.  It is a beautiful, massive lake but we didn’t see very much of the surrounding mountains because of the fog.  We stopped briefly at the bird sanctuary to see the Kaka, the Crested Grebe, and other beautiful birds.  That evening, we had the most amazing 5 star dinner at the Redcliff Café and loved it so much that we booked a table there for the next evening!  Since we were getting up super early the next day to drive to Milford, we went to bed early.

Day 8:  Drive through the Fiordland to Milford Soundnew-zealand1a

This had to be my most favorite day of the entire trip.  Not only was the weather amazing, the entire drive to and from Milford Sound was mind blowing.  The New Zealanders call Milford the 8th Natural Wonder of the World and it is easy to see why.  It is in a remote part of the island and 99% of the Fiordland will never see a human.  The Fiordland has waterfalls aplenty, sweeping, colorful landscapes with millions of gorgeous lupin flowers and gigantic, snow covered mountains.  It truly feels as though you have entered another world.  We left early so we would avoid the dreaded crowds and tour buses that clog the two lane road from 10am-4pm. We stopped constantly as the views just kept getting better and better around every twist and turn.  One notable stop was The Chasm.  The Chasm is where the raging Cleddau River has worn a path straight through the mountain so it virtually disappears into the rock.  There are many massive, perfectly round pothole formations in the limestone where pebbles have worn their way through the rocks.  We were the only two people there at that time of the morning.  It felt majestic just to stand there and listen to the tremendous sound of the powerful river disappearing into the mountain and reappearing on the other side. Continue reading “Exploring the Land of the Silver Fern—Part II”