Dino Protein: New Methods for Old (Very) Samples

Hadrosaurus skeleton vintage engraving.
Hadrosaurus skeleton vintage engraving.

Brachylophosaurus was a mid-sized member of the hadrosaurid family of dinosaurs living about 78 million years ago, and is known from several skeletons and bonebed material from the Judith River Formation of Montana and the Oldman Formation of Alberta. Recent fossil evidence indicates structures similar to blood vessels in location and morphology, have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty  network after degradation of original organic components (i.e., bacterial, slime mold or fungal in origin).  Cleland et al. (1) tested the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence.

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There and Back Again 3: Christchurch, New Zealand (South Island) Travelogue

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the 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 III of the adventures of Mica Zaragoza, a senior client rep, who used his award to travel to Australia and New Zealand.

Helix-Travel-Blog---1-WEBAfter a day of travel, including a quick stop-off in Sydney, we arrived late in New Zealand the evening.  New Zealand is an island nation with strict customs requirements, having been stung in the past by a decision  to import the opossum.

Jumping full into the experience, we rented a car (5-speed) and headed into the City to scope out our hotel ( more on the car later).

During our time in Australia, we had spoken with several travelers who had visited Christchurch for their recommendations.  Without fail, their response was negative, using words like “destroyed” and “rubble” to describe the city.  They referred to a  series of earthquakes in 2011 that crippled the city of Christchurch; the residents were not aware of the risk and structures in the city had not been constructed to withstand such devastation. Continue reading “There and Back Again 3: Christchurch, New Zealand (South Island) Travelogue”

Studying Mitochondrial Fission with NanoBiT Complementation Assay

3D depiction of NanoBiT Protein Complementation

Updated February 15, 2021

If you’re interrogating two proteins to understand the conditions under which they interact, a complementation system enables you to tag each protein. Interaction of the tagged proteins facilitates the complementation of the subunits, resulting in a signal. Here we discuss the NanoBiT complementation assay and describe its use to study mitochondrial fission.

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A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping

Electrophysiologists are talented scientists/artists who see into the events of the cell with amazing detail.
Electrophysiology experiments provide a view into the cell with amazing detail. The paper reviewed here describes a molecular reporter biosensor (NanoBRET) that can offer the same kind of temporal and spatial resolution traditionally reserved for extremely labor-intensive experiments like patch clamp analysis.

I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).

My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.

Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.

So when I read the paper recently published by Mashuo et al. in Science SignalingDistinct profiles of functional discrimination among G proteins determine the action of G protein-coupled receptors”, this sentence really caught my attention:

When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.

They continue:

Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.

A reporter biosensor that can resolve events similarly to patch clamping?!  Amazing. Continue reading “A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping”

Unexpected connections: Gut bacteria influence immunotherapy outcomes

Over the last few years, human microbiome studies have revealed fascinating connections between our colonizing microorganisms and ourselves—including associations between gut bacterial populations and obesity, disease susceptibility, and even mood. The relationship between us and our microbial colonists—once considered completely benign, is now being revealed as an intricate, complicated partnership with the potential to redefine who “we” are in fundamental ways.

Two papers published back-to-back in the November 27 issue of Science add further to this growing body of knowledge—reporting a new and unexpected connection between gut bacterial species and the effectiveness of cancer immunotherapies in mice. The work suggests one reason why such treatments are effective in some circumstances, but not others. Both papers report that the presence of specific bacterial populations may be required for the efficacy of certain treatments, and raise the intriguing question “Could the composition of bacteria in the gut be manipulated to enhance the effectiveness of cancer treatments?”

Continue reading “Unexpected connections: Gut bacteria influence immunotherapy outcomes”

There and Back Again: Part II (Cairns, Australia)

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the 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 Mica Zaragoza, a senior client rep, who used his award to travel to Australia and New Zealand.

Cairns, Australia

24254847-Aussie-Kiwi-1Jumping from down South to way up North, the city of Cairns seems like a touristy beach town, sporting a laid-back feel with lots of cafes, pubs, and restaurants.  The locals drop the hard ‘R’s in most words, making the name of the city hit the ear more like “cans”.  Popping into town, we stopped by the city center and soaking pools along the Esplanade to relax.

With the Esplanade pools right next to the shoreline, I asked one of the locals why no one went swimming in the ocean. The answer was simple: underwater mangroves, mudflats, and saltwater crocs… more on that later. Continue reading “There and Back Again: Part II (Cairns, Australia)”

Worldwide Holiday Celebrations as Told by Promega Employees: Part II

Again, Happy Holidays from Promega employees around the world! We hope you enjoyed the first part of this special holiday blog in which several Promega branch employees shared their country’s holiday traditions. In part II we continue with yet more global traditions and cheer.

Germany
Christian Walczuch, Public Relations for Promega GMBH

In preparation for Weihnachten (Christmas), many families celebrate Advent. This is a time of religious preparation for the 24th, Heiligabend (holy evening or Christmas Eve). Traditional advent activities include the Adventskranz (Advent wreath), which is set up on the 4th Sunday before Christmas Day, or the beginning of the season. Four candles adorn the wreath, and a new one is lit each week. Children also enjoy the advent calendar which contains twenty-four doors (one for each day of December leading up to Christmas). Children open one door each day and find a chocolate treat awaiting them. Many of the calendars also include pictures inside the doors, often Christmas-related.

As in Benelux and Switzerland, a significant part of the Christmas build-up occurs on 6 December, or Nikolaustag, a day commemorating Saint Nicholas. On the evening of 5 December children in Germany place a Nikolausstiefel (a boot or a shoe) in front of the street door. Overnight, the Nikolaus, a figure similar in appearance to Santa Claus in the USA, visits the house and fills the boots with sweets and sometimes even smaller presents if the children were good; otherwise they are left with only a rute (a cane composed of birch twigs).

Christkindlesmarkt_nuernbergDuring the Christmas period, the Weihnachtsmarkt (Christmas market)  becomes a feature of almost every city in the German-speaking countries, town or village, where visitors enjoy stalls, entertainment, and savour food and Glühwein (mulled wine). Famous Christmastime treats include Lebkuchen (gingerbread), Stollen (fruit cake), and Marzipan (confectionery often made into sweets).

The most important day of the season is the 24th where the celebration begins in the afternoon or evening and gifts are exchanged after dinner. One of the most common traditions is for the children to wait to enter into their (locked) living room until a little bell rings. This bell marks the departure of the one delivering gifts, and then the children are free to open them.

  Continue reading “Worldwide Holiday Celebrations as Told by Promega Employees: Part II”

Happy Holidays from Worldwide Promega

Happy Holidays from Promega employees around the world! This holiday season we are excited to share with you the kinds of celebrations we enjoy in the many countries that comprise the Promega family. We hope you feel more connected by reading about the festive customs, foods, symbols and events celebrated in countries that may be far from your own home. Here they are, in the words of our marketing and technical services colleagues.

Please note this is Part I of a two-part blog post. Please look for Part II to be posted on December 25, and have a wonderful holiday!

Italy
Maura Bozzalli, Marketing Manager

Christmas is very important in Italy, not only because we are the country with the Pope, but also, as in the rest of the world, it is a day dedicated to the family and the children. The celebration starts with dinner on Christmas Eve and goes on with the lunch on Christmas Day with gift exchanges as well.

But Christmas is not a real Christmas without a Panettone on the table. Panettone is a special sweet/dessert, not a cake, not a bread, and not anything else – it is Panettone! It has a rounded and very tall cylinder shape (20 cm), and originally it was from the Milan area but now it has spread out all over the country. As is typical of our country, each region has created its own version of the original recipe and of course claims that it is the best one! pannetone

Panettone takes more than two days of work to be done because it requires a long time for rising (10-12 hours) twice. Another typical note about its preparation – after cooking in the oven it is removed with a long fork and placed upside down for at least 5-10 hours until cold to prevent the humidity inside the Panettone from causing it to collapse. That is why it is saved for very special occasions like Christmas, and after so much work and time it is a pleasure to share it with the people you love. Today, not many people make it themselves at home;  instead Panettone is bought in shops but the taste and the tradition is still very lively.

Here all the ingredients needed to make a real Panettone: water, flour, salt, eggs and egg yolk, milk, butter, sugar, candied fruit (orange and citron), raisins, vanilla, and yeast. The official recipe is in Italian, but this tutorial can help you make a Panettone if you want to try for your own very special 2015 Christmas.

Continue reading “Happy Holidays from Worldwide Promega”

ProteaseMAX: A Surfactant for the Most Complex Mixtures

Alternate Proteases Cover

Here we provide two examples of “atypical” experiments that take advantage of the properties of the ProteaseMAX™ Surfactant to improve studies involving digestion of complex protein mixtures.

Example 1
Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. A recent publication (1) illustrated a novel strategy using three different approaches  to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores.

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Top 10 Tips to Improve Your qPCR or RT-qPCR Assays

headache

Scientists have had a love-hate relationship with PCR amplification for decades. Real-time or quantitative PCR (qPCR) can be an amazingly powerful tool, but just like traditional PCR, it can be quite frustrating. There are several parameters that can influence the success of your PCR assay. We’ve highlighted ten things to consider when trying to improve your qPCR results.

Continue reading “Top 10 Tips to Improve Your qPCR or RT-qPCR Assays”