Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal. Continue reading

Pollinator-Plant Interactions, Neanderthal Teeth, Desiccated Tardigrades and Blood Typing: Science News This Week

Keeping up with the pace of scientific discoveries being published each week can be difficult. Here I share a few scientific publications that piqued my interest over the past week:

Pollinators influence evolution of plant traits

Brassica rapa cv. By I, KENPEI [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

Brassica rapa cv. By I, KENPEI [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

To explore the plant-pollinator relationship, researchers studied field mustard, a relative of oilseed rape, under the influence of three pollination conditions: by hand, by bumblebee and by hoverfly. After nine generations, the plants were visually changed. The ones pollinated by bumblebees were taller than the original plant; the ones pollinated by hoverflies, shorter. In addition, the bumblebee-pollinated field mustard developed more fragrant floral compounds and more UV-reflecting petals while the hoverfly-pollinated plants became more self-pollinated. While this experimental was done in isolation from other plants, the research suggests a pollinator can influence the traits evolved by a plant.

Read the Nature Communications research article.

Calculus from Neanderthals reveal diet and probable self-medication

The calcified plaque on teeth of five Neanderthal skulls was scraped, PCR amplified and sequenced to examine what could be learned of diet, behavior and disease. One specimen was eliminated because the DNA did not amplify, one due to environmental contamination, leaving two specimens from Spain and one from Belgium that were used for analysis. The Belgian individual had rhinoceros, sheep and mushrooms caught in its teeth while the Spanish Neanderthals consumed mushrooms, pine nuts, forest moss, and poplar as well as plant fungus. The last two items were of interest because these sequences were found in the Neanderthal suffering from a dental abscess. Poplar contains the active ingredient in aspirin and the fungus was Penicillium from which the first antibiotic was derived. Researchers also compared the bacterial sequences of oral microbes across hominid species and sequenced a draft genome of the 48,000-year-old oral bacterium Methanobrevibacter oralis subsp. neandertalensis.

Read the research article in Nature.

The desiccation tolerance of water bears explained

Scanning electron micrograph of an adult tardigrade (water bear). By Goldstein lab - tardigrades (originally posted to Flickr as water bear) [CC BY-SA 2.0 (http://creativecommons.org/licenses/by-sa/2.0)], via Wikimedia Commons

Adult tardigrade (water bear). By Goldstein lab – tardigrades (originally posted to Flickr as water bear) [CC BY-SA 2.0 (http://creativecommons.org/licenses/by-sa/2.0)], via Wikimedia Commons

The microscopic tardigrades are a creature that inspire microbiologists and others with their cuteness (hence the nickname water bears) and their resilience under dry conditions. However, little was known why they can survive desiccation. New research reveals that unlike other organisms that use sugar to resist drying, tardigrades use disordered proteins to protect itself. These proteins lack stable 3D structures and form glass-like protection under desiccation. Not surprisingly, these proteins are called tardigrade-specific intrinsically disordered proteins or TDPs. By transferring TDPs into yeast, researchers were able to increase yeast tolerance to drying as well as enhance survival.

Read a summary of the research in The Scientist (contains link to research article).

Blood type determined in 30 seconds using a paper-based assay

Matching blood type usually involves centrifuging blood samples to test both red blood cells and plasma, and takes about 30 minutes. However, a rapid test would be useful in emergencies while an alternate test for those without the funds for lab facilities would be beneficial. What about paper infused with dye that could show blood type in seconds, no centrifugation needed? In fact, researchers have developed a paper-based assay that uses microliter volumes of whole blood to determine blood type with a visual indicator. Using immobilized antibodies and a green dye, the blood will clump in the presence of an antibody that is recognized, turning the paper blue to show it has the marker for A (left side of chip) or B (right side of chip). Type AB will have both markers while type O has neither, turning the paper brown on both sides of the chip. Rare blood types and five Rhesus markers can also be analyzed using this paper-based chip assay, starting with a small sample of whole blood.

Read a summary of the research and watch a video of the paper assay chip in Science (contains link to research article).

So NASA Found Some New Exoplanets…Now What?

34412848-March-8-Planets-600x600-WEBYou have probably heard a lot of excitement over NASA’s recent announcement about the discovery of seven earth-size planets found orbiting around the star TRAPPIST-1, which is part of the constellation Aquarius.

These exoplanets are notable because they exist within the habitable zone of the star (nicknamed Goldilocks planets because this area is not too hot and not too cold) and are probably rocky with the potential to contain water on their surface.

A lot of the enthusiasm revolves around the hope that one of these planets might harbor extraterrestrial life or could be suitable for human inhabitants. Of course, many further observations must be made to determine if these scenarios are plausible, not to mention the huge advances in technology that would need to occur so we could actually verify the planetary conditions or send humans 40 light-years away. Continue reading

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed for characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process, reducing the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

Continue reading

Calling All Science PUNdits

As the point of contact for our social media efforts at Promega, I spend a lot of time scanning science-related Twitter, Facebook, Instagram media accounts. There are some science channel managers who do a great job of bringing delight to their followers. Those managers use their platforms to educate—I follow them because they constantly amaze me with new things. I find information that is useful, fun and makes me think “wow, that is interesting.” On my favorite accounts, that new learning comes along with a wry sense of humor, and some of my favorite social media channels are ones that not only teach me new things but do it with a little fun on the side—often in the form of bad science puns.

Promega has the privilege of sponsoring the Cool Science Image contest run by the University of Wisconsin-Madison. Just recently @UWMadScience tweeted about the deadline for the contest, tagging @promega in the tweet. Their tweet included a visual science pun which was not lost on their fellow campus account managers:


That pun started a chain reaction among the other UW accounts that follow @UWMadScience: Continue reading

Biotechnology Youth Apprentice Madhu Gowda Wins GRAND PRIZE at the Capital Science and Engineering Fair

Madhu presents her work.

Madhu presents her work.

Imagine the pleasure Barbara Bielec, the BTC Institute’s K-12 Program Director and co-coordinator of the Dane County Youth Apprenticeship Program in Biotechnology (YAP-Biotechnology), felt when reading this recent message from Sharon Tang, one of our apprentice’s mentors:

“I am unbelievably proud to let you know that Madhu won not only first place for the biological science projects, but also the GRAND PRIZE at the Capital Science and Engineering Fair this weekend! She was at the fair from 7:30am until 4:30pm presenting her work done in our lab and did a fantastic, eloquent job speaking about her project. This was such an impressive honor – she won among over 20 competing students in the region, earned a cash award, and will be competing as a finalist at the Intel international science fair in May. I’m sure she’ll tell you, but I am just over the moon and wanted to share the news as well. Attaching a photo I took of her in action.”

A second year student in the program, Madhu is a senior at Middleton High School. Since November, 2015, she has been working in the lab of Dr. Susan Thibeault in the Department of Surgery, Division of Otolaryngology – Head & Neck Surgery at the University of Wisconsin-Madison. Continue reading

Meet Sue Wigdal, Senior Quality Assurance Scientist on the Spectrum Team

29160613_lPromega will introduce the Spectrum CE System for forensic and paternity analysis. Building this system requires the efforts of many people from many disciplines–from our customers who have told us their needs to the engineers and scientists building the instrument and ensuring its performance. Periodically we will introduce our Promega Connections readers to a team member so that you can have a sneak peak and behind-the-scenes look at Spectrum CE System  and the people who are creating it (of course if you truly want to be the first to know, sign up at www.promega.com/spectrum to receive regular, exclusive updates about Spectrum CE).

Today we introduce Sue Wigdal, Senior Quality Assurance Scientist. Continue reading

Probing RGS:Gα Protein Interactions with NanoBiT Assays

gpcr_in_membrane_on_white2When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases. Continue reading

From Whence You Came, Honey Bee?

Apis melliferaWestern honey bee. By Ivar Leidus (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons

Apis mellifera Western honey bee. By Ivar Leidus (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons

As a new beekeeper, I never really considered much about the origins of honey bees. I knew they were not native to the United States, most are from Europe and the ones that sting in a swarm are called Africanized. Local beekeepers talk about ordering Italians or Carniolans to populate hives, and during a recent local beekeeper’s association presentation, Asian honey bees were mentioned. From where Apis mellifera, the Western honey bee arose, I did not know.

As it turns out, the origin of honey bees is a highly debated topic. Some say they arose from Asia; others say Africa. Recently, researchers from the University of California—Davis used short nucleotide polymorphisms (SNPs) and two sets of previously published whole genome data, included additional sequenced genomes and applied multiple computational methods to analyze honey bee population genetics. They published their conclusions in Genome Biology and Evolution. Continue reading

Restoring Memory in Alzheimer’s Mice with Microbubbles and Ultrasound

Neurons with amyloid plaques.

Neurons with amyloid plaques.

Imagine driving in your car and suddenly not recognizing where you, you don’t remember where you were going and have no idea how to find your way home. What if you looked across the breakfast table at your spouse and no longer recognizing them?  Or maybe you have to brace yourself every time you visit your parent, waiting for the day when they won’t know who you are. This is reality for the estimated 50 million (worldwide) Alzheimer’s suffers and their families.

For a world with an aging population, Alzheimer’s is a growing problem. Recent estimates suggest that 11% of people over the age of 65 have Alzheimer’s disease. For people 85 and older, that number increases to 32% (1).

Alzheimer’s disease is a devastating degenerative brain disease. It is the most common cause of dementia, and is characterized by a decline in cognitive skills such as memory, language skills, communication and problem solving abilities. These symptoms make it difficult for people with Alzheimer’s to perform everyday activities. It also is difficult to diagnose, even more difficult to treat, and, as of now, impossible to cure. Continue reading