Author: Audrey

Happy 47th Birthday to Western Blotting!

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Western Blotting

As we enter a new year for science and continue making new discoveries that change the way we think about and understand science, some aspects are thankfully staying just the same. 2022 marks the 47th birthday of the Western blotting technique! We’re excited to celebrate this tried-and-true method and all the scientific insights it has helped uncover over the years. Do you know who invented the Western blot?

History of the Western Blot

The first mention of “Western blot” was done George Stark’s group at Stanford University in their July 1979 publication, where they used passive transfer of the proteins. This came two years after they first published the first paper using Northern blot. In September of that same year, Harry Towbin published a paper that also used “Western blot” where the group used electrophoretic transfer of proteins instead.

Who invented Western blotting?

Modern day Western blotting was first published by Neal Burnette from Fred Hutchinson Cancer Research Center, published through a paper in 1981. The technique was coined “Western blotting” as a nod to its cousin Southern blotting, with Burnette’s lab being on the west coast. GenHunter has an in-depth analysis of which paper came first that you should check out if you’re interested in learning more.

In either case, Azure Biosystems is thankful to all the brain power that went into creating the Western blot technique and the developments that the scientific community has made along the way.

western blot birthday

What is Western Blotting?

For those who aren’t familiar with the Western blot, it’s an analytical technique used to identify one or more specific proteins in a complex protein mixture. Scientists use it all the time to understand the presence, size, and even the amount of protein in their sample.

>> Interested in this technique for your own research? Learn about the step-by-step process in more detail with our Western Blotting Guidebook.

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!
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How to Image Western Blots

Western blot imaging is usually done in one of two ways: film and digital. Film-based imaging was once the gold standard for imaging but has since been surpassed by digital imaging—thanks to the wider dynamic range, comparably lower costs, and ease of use. Digital imagers and analysis programs two great tools that can help you image Western blots carefully and accurately.

Discover the right Western blotting imaging system for you and your research needs.

Cheers from Azure Biosystems

Whether you’re trying out the Western blotting technique for the first time or it’s been a longstanding part of your research throughout your career, we’ve all got something to cheers for this new year. Azure Biosystems thanks all the scientists who created, developed, and continue to use the Western blot, and we’re excited to celebrate its birthday for many years to come.

Handy Western Blotting Resources

SOURCE
  1. Moritz, CP. 40 years Western blotting: A scientific birthday toast. Journal of Proteomics. Vol 212. 2020.

Sapphire used in a Study that Addresses Sudden Cardiac Death

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Imaging

While not commonly considered a leading cause of death, sudden cardiac death is a health issue that continues to cause premature deaths every year. In fact, sudden cardiac death is estimated to occur over 5 million times around the world every year, and 200,000 of those cases are in the United States alone. 

Because of this, it is imperative the main causes of such events are identified and used to effectively prevent and and lower the general public’s risk of sudden cardiac death. In an effort to help mitigate such risks, Grune et al published a study using an Azure Sapphire to further the scientific community’s knowledge on the subject using both mouse and human models by investigating the relationship between leukocytes and ventricular arrhythmia.

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins.

Research Applications with the Sapphire Biomolecular Imager

Grune et al primarily employed the Azure Sapphire Biomolecular Imager to collect fluorescent images of tissue sections from mouse hearts. The Sapphire is the ideal tool for enhanced quantitative imaging thanks to its stable fluorescent signals, which help achieve reliable quantitation across a wide dynamic range. Its ability to multiplex allows probing for multiple proteins on a single blot, expanding the amount of data that can be collected from each sample.

Leukocytes and Their Role Surrounding Sudden Cardiac Death

The authors sought to identify correlations between leukocytes and ventricular arrhythmia and to also study how hypokalemia (low potassium levels) combined with myocardial infarction may trigger spontaneous ventricular tachycardia.

They established neutrophils incited ventricular arrhythmia in mice and determined a higher circulating neutrophil count was associated with a higher risk of early Vfib and VTand an adverse prognosisin patients with MI.

Using their mouse models, the authors determined a lack of macrophages (which remove dead cells) can impair efferocytosis and lead to a faster death rate in cardiomyocytes (muscles cells that are responsible for making the heart contract).

Post-MI was injected into the mice with an imaging probe that binds to the surface of apoptotic cells and the interior of necrotic cells. The mouse hearts were then processed and imaged with fluorescent reflectance imaging (FRI) on the Sapphire. The imaging probe primarily bound to infarcts and the researchers concluded that absence of macrophages can result in a increased death rate of cardiomyocytes and potential accumulation of these dead cells in mice. 

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Next, the authors investigated the role Mertk, a macrophage receptor that helps mediate efferocytosis and assists with mitochondrial removal from malfunctioning cardiomyocytes. The authors hypothesized that Mertk may help protect against post-MI ventricular arrhythmias like VT and Vfib., as deletion of Mertk receptors leads to lethal arrhythmias in mice with acute MI. Through several small experiments, they concluded that their hypothesis was correct.

Because of these results, the study concludes that macrophages, a type of leukocyte, are indeed effective at averting electrical storms after MI. In addition, macrophages play a large role in defending against post-MI arrhythmias such as VT and Vfib. Lastly, the study’s results demonstrate that unlike macrophages, neutrophils actually increase VT in mice and are closely associated with VT in patients.

This study importantly demonstrates that changes to the function of leukocytes like neutrophils and macrophages may prove to be useful therapeutic tools for reducing the likelihood of sudden cardiac death. A better understanding of these functions may eventually lead to effective anti-arrhythmic drugs.

Used in This Study: Azure Sapphire Biomolecular Imager

The Sapphire is capable of imaging whole specimens, like Arabidopsis thaliana plants, zebrafish, whole mice, and fruit fly, mouse, and frog embryos. For studies on gels or well plates, the Sapphire Biomolecular Imager can be used for in-cell Westerns, chemiluminescence, gel documentation, total protein normalization, bacterial plate imaging, phosphor imaging, and investigating post-translational modifications.

To learn more about the Sapphire Biomolecular Imager used in this study and how it can be an effective tool in your own research, contact us at info@azurebiosystems.com.

The new Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, the Sapphire FL easily adapts to a lab’s changing needs and advancing research.
Learn More about the Sapphire FL

Background on Heart Function and Health Conditions

The myocardium is the muscular layer of the heart that is negatively charged on one side and positively charged on the other. Disruption of the normal rhythmic depolarization of the myocardium can lead to sudden cardiac death. One of the most common causes of sudden cardiac death is thought to be arrhythmias, or abnormal heart rhythms. When arrhythmias occur, the heart is unable to pump blood, which can subsequently lead to heart failure and ultimately death. 

There are two types of arrhythmias: ventricular tachycardia (VT) and ventricular fibrillation (Vfib). They are considered to be some of the most common causes of sudden cardiac death. As such, preventing VT and Vfib may be an effective means of addressing this health concern. Historically, leukocytes have been considered an effective tool in preventing arrhythmias, but they may also contribute to rhythm disorders. In fact, some studies show that health conditions associated with an increased risk of arrhythmia are often also associated with major changes in myocardial leukocyte counts.

Macrophages and neutrophils are types of leukocytes that engulf and digest particles (including pathogens and dead cells) to defend the human body. The process of removing these particles is called efferocytosis. Macrophages are especially responsible for clearing away dead cells; if they do not, the accumulation of cellular debris may indirectly lead to VT. In this study, macrophages are particularly important because they remove dead cardiomyocytes, the muscles cells responsible for making the heart contract, which in turn can help a patient heal after ischemia.

More research done with the Azure Sapphire:

SOURCE

  1. Grune, J. et al (2022). Neutrophils incite and macrophages avert electrical storm after myocardial infarction. Nature Cardiovascular Research, 1(7), 649–664. https://doi.org/10.1038/s44161-022-00094-w

Advancing Bacterium Research with the Azure c400 at Virginia Tech

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Customer Spotlight Imaging Western Blotting

Customer Spotlight: Tam Nguyen, PhD Candidate at Virginia Tech

Since the release of this interview, the Azure c400 has been upgraded to the Azure 400, a flexible fluorescent imager that enables three color fluorescent detection for dyes in the visible range.

Microbiome research has grown exponentially in the last decade, and PhD candidate Tam Nguyen is no stranger to the field. After three years as a molecular biologist and biochemist at Virginia Tech, she has rapidly furthered our current understanding of how microbes may interact with colorectal cancers.

Nguyen is a member of the Slade Lab, headed by Associate Professor Dr. Daniel Slade. The lab’s primary focus is to better understand how a commensal oral bacterium may influence the colorectal tumor microenvironment and induce adverse inflammatory responses in the host. The Slade lab is investigating the host-Fusobacterium interactions and their roles in bacterial pathogenesis and altered host responses in colorectal and pancreatic cancers.

"We like the chemiluminescence application because of its practicality, cost-effectiveness, and easy usage,"

Tam Nguyen

Nguyen and the lab have made great strides in understanding how Fusobacterium nucleatum, an opportunistic oral pathogen that has garnered increasing attention, interacts with colorectal cancer cells. The Slade Lab is one of the few labs with the capacity to make genetic modifications in Fusobacterium nucleatum, making them a great resource to help move the field forward to better expand on the topic of tumor microbiome. Through her years at Virginia Tech, Nguyen has helped uncover how Fusobacterium can establish invasion and long-term survival.

Azure 400 Visible fluorescent imaging system
The Azure 400 is capable of three-channel visible fluorescence detection, which enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes. This fluorescent imager allows users to simultaneously image and quantify up to three different targets.
Learn More about the Azure 400
Tam Nguyen with Azure c300
Nguyen, pictured with Western blot results on the lab's Azure c400 Imager

To begin her research, Nguyen cultures F. nucleatum statically in an anaerobic chamber to mimic the living condition of this bacterium since it resides in oxygen-free pockets in the mouth. To focus on bacterial intracellular survival, Nguyen extracts protein lysates from the bacteria once they are at certain growth characteristics, and performs Western blot analysis followed by visualization with the Azure c400 Imaging System.

Nguyen regularly utilizes this approach to understand the differences in protein expression among bacterial strains that have been genetically modified. She grows her bacteria in an anaerobic chamber with the appropriate gas mixture but skips the shaking step in the incubation period due to its non-motile nature. “We mainly use the instrument for Western blot analysis, which is routinely used in our lab for analyzing protein expression,” confirms Nguyen.

Nguyen will be defending her research in a public seminar next month with the help of the publication-worthy analysis from the Azure c400 Imager. She looks forward to how her work may influence future cancer microbiome studies and how the Slade Lab’s work can help close our knowledge gap on understanding disease-centric relationships between biological systems and microbes.

Together, Nguyen and the Slade Lab team will continue to use the Azure c400 Imager in their recent discoveries in an effort to eliminate F. nucleatum to combat disease. Their research helps to develop more effective cancer treatment methods.

For more information on the Slade Lab and Dr. Slade’s research at Virginia Tech, check out their lab’s website.

Ready to learn more about how easy Western blotting is by using an Azure Imager?

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Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager

North Carolina’s Elite Christmas Tree Industry

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Customer Spotlight Imaging Western Blotting

Customer Spotlight: Adarsha Devihalli, PhD Candidate at North Carolina State

Since the release of this interview, the Azure c400 has been upgraded to the Azure 400, a flexible fluorescent imager that enables three color fluorescent detection for dyes in the visible range.

Nestled in the southern region of the Appalachian Mountains is an environmentally beneficial abundance of Fraser fir—the most sought-after Christmas tree in the USA. Thanks to its charming aroma, soft and durable needles, and eye-catching silhouette the tree forms the foundation of a multi-million dollar industry in North Carolina. It is these qualities combined with this unique geography that make North Carolina the second-leading Christmas tree producer in the United States. And while Fraser firs are heavily popular with holiday enthusiasts, they’re also extremely vulnerable to Phytophthora, a common cause of root rot disease.

"The Azure 400 Imager comes in [and is] a multi-user instrument…so we don’t have to run different instruments or look for labs that have all the instruments for us. Once I’m sure I’ve identified Phytophthora, I can use the cultures for my downstream experiments.”

Adarsha Devihalli
Adarsha and Dr. Whitehill standing next to azure c300
Adarsha and Dr. Whitehill with Azure 400 imaging system in their lab.

Several scientists at North Carolina State University are not letting this pathogen get in the way of Christmas tree production. For PhD student Adarsha Devihalli, the solution is in the molecular details. His research focuses on studying a particular strain of Phytophthora and its genetic code. His initial work focused on pathogen identification. In this next phase of his research, he will use functional genomics tools to enable the identification of genes in the pathogen important for the initiation of the infection process.

Devihalli isn’t the only one working on Phytophthora, either. He is a member of the Christmas Tree Genetics (CTG) Program, headed by Dr. Justin G. A. Whitehill, Assistant Professor and Director of the Christmas Tree Genetics Program at NC State University.

Under the guidance of Dr. Whitehill, Devihalli is studying this devastating disease to better understand the issues at hand. Together, Whitehill CTG lab members are working towards the development of novel genomic resources for Fraser fir to combat several pests of these celebrated trees.

Azure 400 Visible fluorescent imaging system
The Azure 400 is capable of three-channel visible fluorescence detection, which enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes. This fluorescent imager allows users to simultaneously image and quantify up to three different targets.
Learn More about the Azure 400

How the samples are collected

To begin his experimental process, Devihalli first visits the NC Department of Agriculture’s research station in Ashe County – located approximately four hours away from the university in Raleigh. He looks for disease-related symptoms on Fraser firs, collects samples, and returns to the lab for culturing, identification, and analysis using the Azure 400 Imaging System.

Looking to the future

Together, the Whitehill CTG lab and Devihalli intend to use their experimental results to help further current knowledge of the Fraser fir genome, and uncover potential genetic resistance mechanisms to Phytophthora root rot.  Ultimately, they plan to develop better mitigation methods for root rot in the country’s most beloved Christmas tree.

“At present, there is no publicly available sequencing information for these species,” explains Devihalli. “We don’t have a genome sequence for Fraser fir, so this is a big goal for our lab [yet].”

DISCOVER: Azure 400 Imager

For more information on Dr. Whitehill’s Christmas tree research at NC State, visit https://research.cnr.ncsu.edu/sites/whitehilllab/