Tips and Tools

From Hit to Live-Cell Target Engagement Assay: DNA-Encoded Library Screens and NanoBRET™ Dye

Posted on by Tian Yang

Monitoring and quantifying drug-target binding in a live-cell setting is important to bridging the gap between in vitro assay results and the phenotypic outcome, and therefore represents a crucial step in target validation and drug development (1). The NanoBRET™ Target Engagement (TE) assay is a biophysical technique that enables quantitative assessment of small molecule-target protein binding in live cells. This live-cell target engagement assay uses the bioluminescence resonance energy transfer (BRET) from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent tracer that reversibly binds the target protein of interest. In the presence of unlabeled test compound that engages the target protein, the tracer is displaced, and a loss of BRET signal is observed. Due to the tight distance constraints for BRET, the signal measured is specific to the target fused to NanoLuc® luciferase.

Live-cell target engagement assay using NanoBRET to measure small molecule binding to a target transmembrane protein.

Promega offers over 400 ready-to-use assays for multiple target classes, including kinases, E3 ligases, RAS, and many others. For targets that do not have an existing NanoBRET™ TE assay, Promega offers NanoBRET™ dyes, NanoLuc® cloning vectors, and NanoBRET™ detection reagents to develop novel NanoBRET™ TE assays.

To learn more about the NanoBRET™ TE platform, see the NanoBRET™ Target Engagement Technology Page on our website.

One critical component in the development of novel NanoBRET™ TE assay is the creation of the cell-permeable fluorescent tracers (NanoBRET™ tracers) against the target protein of interest. The tracers are bifunctional, consisting of a NanoBRET™-compatible fluorophore and a target-binding moiety connected by a linker. While the NanoBRET™ 590 dyes have demonstrated consistently robust cell permeability and optimal spectral overlap with NanoLuc® for BRET, a ligand capable of binding to the target protein of interest needs to be identified to generate a NanoBRET™ tracer.

What Are DNA-Encoded Libraries?

DNA-Encoded Libraries, (DELs), have emerged as powerful tools for discovering small molecule ligands to target proteins of interest at an unprecedented scale. . owing to the ability of a DEL  to enable the synthesis of larger libraries of compounds and to target proteins without any prior structural knowledge of the proteins or their ligands (2). Because each member of a DEL contains a DNA barcode and a small molecule separated by a linker, DEL is primed for discovering leads within therapeutic modalities that rely on bifunctional chemistry, such as proteolysis targeting chimeras (PROTACs). Since NanoBRET™ tracers are also bifunctional, ligands identified from DEL selections could serve as ideal candidates for developing novel NanoBRET™ tracers that can enable NanoBRET™ TE assays for new targets.

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Insects and Science: Optimizing Work with Sf9 Insect Cells

Posted on by Shannon Sindermann

Insects are a keystone species in the animal kingdom, often providing invaluable benefits to terrestrial ecosystems and useful services to mankind. While many of them are seen as pests (think mosquitos), others are important for pollination, waste management, and even scientific research.

Insect biotechnology, or the use of insect-derived molecules and cells to develop products, is applied in a diverse set of scientific fields including agricultural, industrial, and medical biotechnology. Insect cells have been central to many scientific advances, being utilized in recombinant protein, baculovirus, and vaccine and viral pesticide production, among other applications (5).

Therefore, as the use of insect cells becomes more widespread, understanding how they are produced, their research applications, and the scientific products that can be used with them is crucial to fostering further scientific advancements.

Primary Cell Cultures and Cell Lines

Cell culture - Cell lines - Insect Cells

In general, experimentation with individual cells, rather than full animal models, is advantageous due to improved reproducibility, decreased space requirements, less ethical concerns, and a reduction in expense. This makes primary cell cultures and cell lines essential contributors to basic scientific research.

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No Horsin’ around with Halal Meat Authentication

Posted on by Promega

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.  

People’s diets are frequently influenced by a wide range of variables; with environment, socioeconomic status, religion, and culture being a few of the key influencers. The Muslim community serves as one illustration of how culture and religion can hold influence over people’s eating habits.

Halal meat on cutting board

Muslims, who adhere to Islamic teachings derived from the Qur’an, frequently base dietary choices on a food’s halal status, whether it is permissible to consume, or haram status, forbidden to consume. With the population of Muslims expected to expand from 1.6 billion in 2010 to 2.2 billion by 2030, the demand for halal products is anticipated to surge (2).

By 2030, the global halal meat market is projected to reach over $300 billion dollars, with Asia-Pacific and the Middle East regions being the largest consumers and producers of halal meat products (3). Furthermore, increasing awareness and popularity of halal meat among non-Muslim consumers, as well as strengthening preference for ethical and high-quality meat, are all contributing to demand.  

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Streamlining Disease Diagnostics to Protect Potato Crops

Posted on by AnnaKay Kruger
A potato farmer holds a handful of potatoes. Scientists are working to protect potato crops from disease.
The WSPCP works to provide seed potato growers with healthy planting stock

The mighty potato—the Midwest’s root vegetable of choice—is susceptible to a variety of diseases that, without proper safeguards, can spell doom for your favorite side dishes. Founded in 1913 and housed in the Department of Plant Pathology at the University of Wisconsin-Madison, the Wisconsin Seed Potato Certification Program (WSPCP) helps Wisconsin seed potato growers maintain healthy, profitable potato crops year-to-year through routine field inspections, a post-harvest grow-out and laboratory testing.

While WSPCP conducts visual inspections for various seed potato pathogens, their diagnostic laboratory testing is primarily focused on viruses such as Potato virus Y (PVY), which can cause yield reduction and tuber defects, along with select bacteria such as Dickeya and Pectobacterium species that cause symptoms like wilting, stem rot and tuber decay.

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Custom Manufacturing: Translating Research into Product

Posted on by Natalie Larsen

Scientists around the world are focusing their energy and resources on translating advances made in clinical research into relevant biotechnology, clinical, and applied products that improve our health and well-being. Once research looks promising, there is substantial pressure to expedite the release of that product or assay in the market.

For many organizations focused on developing these advanced products, their expertise and core competencies are in developing the assay. Often, they do not have the experience, infrastructure, or quality systems in place to support large-scale production, packaging, or distribution of their newly developed assay in a way that is also in compliance with relevant regulatory requirements. These next steps become a barrier to realizing the value of the research. Working with a custom or contract manufacturing partner can lower this barrier and expedite the time to market.

Custom Manufacturing

Be careful not to confuse custom manufacturing with original equipment manufacturer (OEM) products. OEM products are existing products from one company that another company rebrands and sells. Custom manufacturers typically focus on providing more comprehensive services that can be adapted to produce a new product. Custom manufacturing is not “one size fits all” and can be simple or complex, such as producing a single component to a final finished product.

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Have No Fear, qPCR Is Here: How qPCR can help identify food contamination

Posted on by Shannon Sindermann

Foodborne disease affects almost 1 in 10 people around the world annually, and continuously presents a serious public health issue (9).

Food Contamination-Strawberries-Blueberries-Magnifying glass
Food Contamination is common and can be seen in a variety of forms and food products.

More than 200 diseases have evolved from consuming food contaminated by bacteria, viruses, parasites, and chemical substances, resulting in extensive increases in global disease and mortality rates (9). With this, foodborne pathogens cause a major strain on health-care systems; as these diseases induce a variety of different illnesses characterized by a multitude of symptoms including gastrointestinal, neurological, gynecological, and immunological (9,2).

But why is food contamination increasing?

New challenges, in addition to established food contamination hazards, only serve to compound and increase food contamination risks. Food is vulnerable to contamination at any point between farm and table—during production, processing, delivery, or preparation. Here are a few possible causes of contamination at each point in the chain (2):

  • Production: Infected animal biproducts, acquired toxins from predation and consumption of other sick animals, or pollutants of water, soil, and/or air.
  • Processing: Contaminated water for cleaning or ice. Germs on animals or on the production line.
  • Delivery: Bacterial growth due to uncontrolled temperatures or unclean mode of transport.
  • Preparation: Raw food contamination, cross-contamination, unclean work environments, or sick people near food.

Further emerging challenges include, more complex food movement, a consequence of changes in production and supply of imported food and international trade. This generates more contamination opportunities and transports infected products to other countries and consumers. Conjointly, changes in consumer preferences, and emerging bacteria, toxins, and antimicrobial resistance evolve, and are constantly changing the game for food contamination (1,9).

Hence, versatile tests that can identify foodborne illnesses in a rapid, versatile, and reliable way, are top priority.

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Scaling Up to Measure 40,000 Data Points a Day with GloMax® Microplate Readers

Posted on by Jordan Nutting

Traditional approaches for protein degrader compound screening like Western blotting can be laborious, time consuming and cannot be streamlined with automation. By implementing a high-throughput, automated workflow that uses our CRISPER/Cas9 knock-in cell lines, live-cell bioluminescent assays and sensitive GloMax® Discover microplate readers, our custom assay services offer protein degradation profiling at an accelerated rate.  

To do this, we collaborated with HighRes® Biosolutions, to develop an automated system that can screen up to 100 384-well plates each day, generating roughly 40,000 data points with minimal hands-on work.

Learn how bioluminescent tools like HiBiT and NanoBRET™ technology can help you answer key questions in your targeted protein degradation research.

An important step of building this system is to integrate four GloMax® Discover microplate readers into the automated system using instrument’s built-in SiLA2 communication driver. The driver software makes it easy to connect the microplate readers with HighRes® Biosolution’s robotic components.

Check out our setup in the video below.

See how we’ve integrated GloMax® Discover microplate readers into a high-throughput automated system for profiling protein degraders in live cells.
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It’s Time to Automate Your Plasmid Purification

Posted on by Jordan Nutting

In the fifty years since the first reported transformation of recombinant plasmids into bacteria (1), plasmid cloning has become one of the pillars of synthetic biology research and manufacturing biopharmaceuticals.

But purifying plasmids is no small feat. It can often take hours of hands-on time to go from culture to eluate with low-throughput and time-sensitive manual methods. Automating plasmid purification is the way to go, whether you’re isolating a single plasmid from a large volume culture or creating a library of thousands of different constructs.

Working in a biosafety cabinet filled with flasks and culture plates containing yellow bacterial cultures, a researcher harvests a culture.
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Avoid the Cloning Blues This Season

Posted on by Michele Arduengo

I was blasting a holiday music playlist while driving recently, and Presley’s Blue Christmas played. I couldn’t get the phrase “Christmas Cloning Blues” out of my mind, and by the time I arrived at my destination, this happened:

Cloning Blues Christmas

(to the tune of Blue Christmas by Elvis Presley)

Blue and white colonies on a selective plate. Careful planning can help you avoid the cloning blues
Blue/White cloning is a standard technique in molecular biology labs.

I’ll have a blue Christmas without you

Colonies so blue, insert without you

Incubating my plates at 37 degrees

Won’t be the same if you’re not in lacZ

And all those blue colonies are forming

When my lab mates’ clonings are performing

They’ll be doing alright,

With their plates all filled with white

But I’ll have a blue, blue, blue cloning

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What’s New with Promega Resources?

Posted on by Riley Bell

Whether you’re looking to learn about nucleic acid analysis, wondering how to do an ELISA assay, or simply have ten minutes to kill in your day, Promega resources have you covered! We’ve revamped and upgraded our hub of self-service resources so we can provide the most up-to-date scientific support. Here are some highlights of what’s new:

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