Wetlands, Water Quality and Rapid Assays

toad

The storms of the previous day had moved eastward, leaving in their wake flooded farm fields and saturated roadside wetlands. At dusk, we loaded the Ford Escort wagon and headed south. We bumped along the maze of farm roads intent upon listening for croaks and snores in the night. At one roadside wetland, I heard my first congress of Spadefoot toads. The sound was deafening, invoking everything that a “congress of snoring toads” brings to mind. Around the corner, in a low spot of a corn field, a lone Spadefoot toad called for a mate; he was joined by a rather enthusiastic Copes Gray tree frog and several chorus frogs. The congress down the road provided a rolling bass to these more melodic anurans.

Wetlands exist in many different shapes and sizes and in many different geographies: coastal margins, mountain valleys, beaches and rocky shores, estuarine wetlands where tidal saltwater and freshwater mix, and inland wetlands. Some of them are ephemeral, some of them permanent. Wetlands serve many different functions, from providing habitat and food for plants and animals to offering protection from floods and maintaining water quality. One acre of one-foot deep wetland is estimated to hold 330,000 gallons of water. Coastal wetlands are important for reducing storm erosion by decreasing tidal surge and buffering the wind. In the US alone, this benefit has an estimated value of $23.2 billion dollars each year. Continue reading “Wetlands, Water Quality and Rapid Assays”

Executing a NanoBRET™ Experiment: From Start to Data

This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Glycobiology Research and Training Opportunities are Plentiful

glycans on cell surface
Artist’s rendering of asymmetrically-branched carbohydrates on cell surface proteins.

Glycobiology is the study of glycans, the carbohydrate molecules that cover the surface of most human cells. Glycans attach to cell surface proteins and lipids, in a process called glycosylation. These cell surface structures are responsible for processes as varied at protein folding, cell signaling and cell-cell recognition, including sperm-egg recognition and immune cell interactions. Glycans play important roles in the red blood cell antigens that distinguish blood types O, A and B.

Opportunities in Glycomics Research
As more is learned about the role of glycans in cell communication, they are becoming important disease research targets, particularly the role of glycans in cancer and inflammatory diseases (2).

Some of the open questions surrounding glycans and glycosylation include glycan structural diversity. While some carbohydrates exist as straight or symmetrically branched chains, those populating the human glycome are asymmetrically branched, making them difficult to create and study in the laboratory (3). Continue reading “Glycobiology Research and Training Opportunities are Plentiful”

What Could You Do with a Faster, More Consistent ADCC Reporter Bioassay?

Fc receptor-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) by which antibodies target diseased cells for elimination. Traditional methods for measuring ADCC require primary donor peripheral blood mononuclear cells (PBMCs) or purified natural killer (NK) cells that express Fc receptors on the cell surface. Killing of target cells is an endpoint of this pathway activation and is used in classic ADCC bioassays.

PBMCs and NK cells are notoriously difficult to isolate and culture. Furthermore, cultured cells can be a source of variability.

There is a Better Way

Watch this video to learn why traditional ADCC assays can be problematic. You’ll also learn a solution. Find out how  to not only save time but also reduce assay variability.

For more details on the benefits of working with ADCC Reporter Bioassays go to the product page.

There you’ll see how standardized reagents in Promega ADCC Reporter Bioassays ensure better results and better consistency in an ADCC Reporter Bioassay that saves you time.

Quantitating Kinase-Inhibitor Interactions in Live Cells

Kinase target engagement is a new way to study kinase inhibitors for target selectivity, potency and residency. The NanoBRET™ TE Intracellular Kinase Assays enable you to quantitate kinase-inhibitor binding in live cells, making these assays an exciting new tool for kinase drug discovery research.

For today’s blog about NanoBRET™ TE Intracellular Kinase Assay, we feature spokesperson Dr. Matt Robers. Matt is part of Promega’s R & D department and is one of the developers of the NanoBRET™ TE Intracellular Kinase Assay. Continue reading “Quantitating Kinase-Inhibitor Interactions in Live Cells”

From Drug Screening to Agriculture to Cardiac Development, Dual Luciferase Reporters Bring You the Story

Today’s blog was written by guest blogger Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

Reporter gene assays have been critical for the study of a wide-range of biological questions, from regulation of gene expression to cellular signaling. While reporter gene assays constitute a large group of technologies, here we highlight the diversity of new discoveries enabled by highly quantitative and easily measured bioluminescent luciferase-based reporter assays. Below are our top picks of exciting research discoveries involving the Dual-Luciferase Reporter Assay format using firefly and Renilla luciferases. Continue reading “From Drug Screening to Agriculture to Cardiac Development, Dual Luciferase Reporters Bring You the Story”

All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.
With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA like that induced by CRISPR-Cas9 is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”

Millions of Pickles, Pickles in the Sea

For a few years beginning late in 2013, warmer ocean conditions in the eastern Pacific prompted the appearance of unexpected species and toxic algal blooms that devastated others. When temperatures cooled in 2017, the marine ecosystems seemed to be returning to normal. Except for the pyrosomes. Although these previously rare organisms did start to wash up on beaches during the periods of warming, they began to appear by the millions from Oregon to Alaska that spring.

Pyrosomes
Photo by Steven Grace.

Some combination of ideal conditions led pyrosomes to multiply, dominate the ocean surface and wash up on beaches along the US and Canadian Pacific Coasts. Pyrosomes typically exist offshore, far below the surface in warm, tropical waters all over the world. Their sudden proliferation in other areas is likely due to the warm, Pacific ocean “blob,” although atypical sea currents and changes in pyrosome diet have been offered as other possible explanations.

While the appearance of pyrosomes impeded the efforts of fisherman by clogging nets and filling hooks, greater ecological effects have yet to be observed. As we celebrate World Oceans Month, pyrosomes offer a mesmerizing example of the astounding biological diversity our oceans have to offer and, perhaps, a cautionary tale of the impact climate change can have on those marine lifeforms.

The pyrosome species common in the NE Pacific, Pyrosoma atlanticum, goes by a few other colorful names. Each name reveals something captivating about these creatures. Commonly called “sea pickles” due their size, shape and bumpy texture (like a transparent cucumber), these are not single organisms, but colonies formed by hundreds or thousands of individual multicellular animals call zooids.

Continue reading “Millions of Pickles, Pickles in the Sea”

Kinase Drug R & D: Helping Your Inhibitor Make the Cut

Finding the best inhibitor for your kinase doesn’t have to be a long trip.

A recent paper in Journal of Medicinal Chemistry, “Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development” (1) details some elegant work in chemical modification and extensive testing during exploration of inhibitors for BTK. As a warmup to the article, here is a brief BTK backstory.

BTK (Bruton Tyrosine Kinase): Importance in Health and Disease 

Bruton’s tyrosine kinase (BTK) was initially identified as a mediator of B-cell receptor signaling in the development and functioning of adaptive immunity. More recent and growing evidence supports an additional role for BTK in mononuclear cells of the innate immune system, especially dendritic cells and macrophages. For example, BTK functions in receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. BTK has recently been identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome (2). Continue reading “Kinase Drug R & D: Helping Your Inhibitor Make the Cut”

ViaFect™ Reagent: Building Assays in Difficult Cells

Transfection can sometimes seem more like an art than a science—the perfect transfection experiment being dependent on optimization of conditions, including cell density, transfection reagent and DNA:reagent ratio. No one reagent is perfect for every cell type, so there is the added challenge of optimizing performance in your cell line of choice—which may fall into the well-populated “difficult-to-transfect” category  that includes many primary cells.

Among transfection reagents, Lipofectamine® (Thermofisher), and FuGENE® (Promega) are popular and widely used choices. Viafect™ Transfection Reagent is newer and less well-known, but gaining popularity as a high-performance, low-toxicity reagent that performs well across a wide range of cell lines. In head-to-head comparisons with FuGENE and Lipofectamine, Viafect outperformed or equaled the others for expression of transfected reporter genes and resulting cell viability (see the data in this article).

The story of ViaFect begins with Promega Custom Assay Services (CAS), a group that uses Promega technologies to construct made-to-order assays, typically in a cell line. Many projects from the CAS group involve transfecting cells with expression vectors and reporter vectors. In some instances, customers contact CAS to have an assay constructed in a difficult cell line, after attempting and failing, or experiencing difficulty building the assay themselves.

CAS projects start with a proof-of-concept experiment using transient transfection before moving on to production of a clonal, stable cell line. For difficult cell lines, the CAS group previously turned to electroporation after exhausting lipid-based transfection options. Electroporation often worked, but success came with a price—cytotoxicity. The CAS group challenged R&D to find a better solution—better transfection with low toxicity for difficult-to-use cells. The result of that challenge is the ViaFect™ Transfection Reagent. Continue reading “ViaFect™ Reagent: Building Assays in Difficult Cells”