Meet the Mighty Masked Masters of Measurement

Scientific investigation is an iterative process, for which reproducibility is key. Reproducibility, in turn, requires accuracy and precision—particularly in measurement. The unsung superheroes of accuracy and precision in the research lab are the members of your local Metrology Department. According to Promega Senior Metrologist, Keela Sniadach, it’s good when the metrology department remains unsung and behind the scenes because that means everything is working properly.

Holy Pipettes, Scientists! We have a metrology department?! Wait…what’s metrology again?

Callibration technician checks out a multipipettorMetrology (the scientific study of measurement) got its start in France, when it was proposed that an international length standard be based on a natural source. It was from this start that the International System of Units (SI), the modern metric system of measurement, was born.

Metrology even has its own day: May 20, which is the anniversary of the day that the International Bureau of Weights and Measures (BIPM) was created by the Meter Convention in Paris in 1875. The job of BIPM is to ensure worldwide standards of measurement.

For life scientists, metrology centers around making sure the equipment used everyday—from pipettes to heating blocks to centrifuges—is calibrated and measuring correctly. Continue reading “Meet the Mighty Masked Masters of Measurement”

Curiosity and Collaboration: A PhD Journey

Concepcion Sanchez-Cid didn’t know she wanted to be a scientist when she was older. She grew up with a love of music and played the violin, but her curiosity and eagerness to learn drove her down the path for a career in biomedical research.

Hear more of Concepcion’s story:

 

As a Master’s student at the University of Granada, Concepcion studied biotechnology and landed an internship at the Promega Europe Training and Application Lab (PETAL) in France. She worked with the Applications Team to develop protocols for DNA and RNA extraction from soil. When she decided to pursue a PhD, she received a sponsorship from Promega and enrolled as a student at the University of Lyon while also remaining an employee at PETAL.

Concepcion says that the balance between both worlds—academia and industry—provide her with technical skills and a unique support network that has helped shape her PhD thesis work. “Working at a university and a company at the same time…you get very different feedback from people that are very specialized, and they really know what they’re doing, so at the end you integrate everything,” she says. “It’s one of the things I appreciate most about my PhD.” Continue reading “Curiosity and Collaboration: A PhD Journey”

Activating the Inflammasome: A New Tool Brings New Understanding

Innate immunity, the first line of immune defense, uses a system of host pattern recognition receptors (PRRs) to recognize signals of “danger” including invariant pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These signals in turn recruit and assemble protein complexes called inflammasomes, resulting in the activation of caspase-1, the processing and release of the pro-inflammatory cytokines IL-1ß and IL-18, and the induction of programmed, lytic cell death known as pyroptosis.

Innate immunity and the activity of the inflammasome are critical for successful immunity against a myriad of environmental pathogens. However dysregulation of inflammasome activity is associated with many inflammatory diseases including type 2 diabetes, obesity-induced asthma, and insulin resistance. Recently, aberrant NLRP3 inflammasome activity also has been associated with age-related macular degeneration and Alzheimer disease. Understanding the players and regulators involved in inflammasome activity and regulation may provide additional therapeutic targets for these diseases.

Currently inflammasome activation is monitored using antibody-based techniques such as Western blotting or ELISA’s to detect processed caspase-1 or processed IL-1ß. These techniques are tedious and are only indirect measures of caspase activity. Further, gaining information about kinetics—relating inflammasome assembly, caspase-1 activation and pyroptosis in time—is very difficult using these methods. O’Brien et al. describe a one-step, high-throughput method that enables the direct measurement of caspase-1 activity. The assay can be multiplexed with a fluorescent viability assay, providing information about the timing of cell death and caspase-1 activity from the same sample. Continue reading “Activating the Inflammasome: A New Tool Brings New Understanding”

B Cells, T Cells and Now X Cells?

The cause of type 1 diabetes (T1D) is not well understood. What is known is that in T1D, immune cells attack pancreatic islet cells that produce insulin. In addition, insulin is an autoantigen that activates T cells in diabetic persons.

A new discovery by Ahmed et al. could further T1D understanding. These findings are also setting B and T cell paradigms on their ear.

About B Cells and T Cells

Components of the B cell receptor.
Components of the B-cell receptor.Image by CNX OpenStax. Used with permission under Wikimedia Commons.

B cells (B lymphocytes) are part of the cellular immune response. They act by means of surface receptor molecules that are immunoglobulins. These B cell receptors are created by highly variable gene rearrangements that result in a huge variety of these surface immunoglobulin molecules. The beauty of B cell receptors (BCR) lies in the fact that, through random gene rearrangements comes a such large variety of B cell surface receptors, that any foreign antigen that makes its way into the body is recognized and snagged by a B cell receptor.

B cells then internalize, process and present these antigens to T cells. Continue reading “B Cells, T Cells and Now X Cells?”

Lighting Up GPCR Research with Bioluminescent Tagging

G Protein-Coupled Receptors (GPCRs) are a very large, diverse family of transmembrane receptors in eukaryotes. These receptors detect molecules outside the cell and activate internal signaling pathways by coupling with G proteins. Once a GPCR is activated, β-arrestins translocate to the cell membrane and bind to the occupied receptor, uncoupling it from G proteins and promoting its internalization.

Reporter tags are useful for studying the dynamics of GPCRs and associated proteins, but large tags can disrupt the receptors’ native functioning, and often overexpression of the tagged protein is required to obtain sufficient signal. Here is one example of how researchers have used the small, bright NanoLuc® luciferase to overcome these common challenges and answer questions about GPCRs. Continue reading “Lighting Up GPCR Research with Bioluminescent Tagging”

Bottom-up Proteomics: Need Help?

The use of mass spectrometry for the characterization of individual or complex protein samples continues to be one of the fastest growing fields in the life science market.

Bottom-up proteomics is the traditional approach to address these questions. Optimization of each the individual steps (e.g. sample prep, digestion and instrument performance) is critical to the overall success of the entire experiment.

To address issues that may arise in your experimental design, Promega has developed unique tools and complementary webinars to help you along the way.

Here you can find a summary of individual webinars for the following topics: Continue reading “Bottom-up Proteomics: Need Help?”

Why You Don’t Need to Select a Wavelength for a Luciferase Assay

It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.

Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest. Continue reading “Why You Don’t Need to Select a Wavelength for a Luciferase Assay”

The Simplex Things In Life: Utilizing Artificial Intelligence Models to Better Understand Autism

Autism Spectrum Disorder, or ASD, is nothing if not unique.

The way ASD manifests itself in people is unique; although it most often presents as some form of variable impairment in social interaction and communication, each individual has behaviors and habits that are as unique to them as snowflakes are to one another.

ASD has also proven itself to be a uniquely challenging disorder to study. In the past decade, de novo (new) mutations have been identified as key contributors to causality of ASD. However, the majority of these identified de novo mutations are located in protein-coding genes, which comprise only 1–2% of the entire human genome.

Up to this point, a majority of previous research has focused on identifying mutations located in the 20,000 identified genes in the protein-coding region, which would seem like a promising approach. Genes are the genetic blueprints for creating proteins, which control and perform crucial tasks in our bodies, such as fighting off infections, communicating between your organs, tissues, and cells as chemical messengers, and regulating your blood sugar levels. It seems like basic math: Genes + Mutations = Mutated Proteins. Mutated Proteins = Disrupted Protein Function.

However, it has been observed that all the known genes that are ASD-associated can explain only a minor fraction of new autism cases, and it is estimated that known de novo mutations in the protein-coding region contribute to not more than 30% of cases for individuals who have no family history of autism (better known as simplex ASD). This provides evidence to suggest mutations contributing to autism must additionally occur elsewhere in the genome. Continue reading “The Simplex Things In Life: Utilizing Artificial Intelligence Models to Better Understand Autism”

When Proteins Get Together: Shedding (Blue) Light on Cellular LOV

NanoBRETNo protein is an island. Within a cell, protein-protein interactions (PPIs) are involved in highly regulated and specific pathways that control gene expression and cell signaling. The disruption of PPIs can lead to a variety of disease states, including cancer.

Two general approaches are commonly used to study PPIs. Real-time assays measure PPI activity in live cells using fluorescent or luminescent tags. A second approach includes methods that measure a specific PPI “after the fact”; popular examples include a reporter system, such as the classic yeast two-hybrid system.

Continue reading “When Proteins Get Together: Shedding (Blue) Light on Cellular LOV”

Research-Based Training for Sustainable Use and Management of Marine Ecosystems in Namibia

In my science blog research/writing, news reports are usually pulled from US sources. But interesting scientific research is obviously being conducted in many places around the globe. When this story from Namibia came along, there was so much I didn’t know. It was time to catch up.

relief map of Namibia
Relief map of Namibia. Image by Natural Earth and Kbh3rd with permission under Wikimedia commons.

Namibia is Exactly Where in Africa?

Namibia is one of the world’s youngest countries, having gained independence from South Africa in 1990. Situated northwest of the country of South Africa on the Atlantic Ocean, Namibia is arid, composed largely of desert.

This blog is about research conducted at the Sam Nujoma Research Center, University of Namibia, on Henties Bay. Henties Bay (not shown on this map) is in the region of Erongo, located in the center of Namibia along the coast. Henties Bay has become a tourist destination in part due to the abundance of fish and marine life found there.

Sam Nujoma Research center.
The Sam Nujoma Research Center of University of Namibia, located near Henties Bay.

Continue reading “Research-Based Training for Sustainable Use and Management of Marine Ecosystems in Namibia”