c600 Used in a Study to Research Malaria Transmission in Mosquitoes

Categories
Immunoassay Western Blotting

Malaria is a mosquito-borne disease that infects millions of people every year and kills hundreds of thousands of those infected. The Plasmodium parasites are the cause of malaria and are transmitted through the bite of an infected Anopheles mosquito.

This malaria research study from NIH highlights the possibility of expressing other human proteins from the fibrinolytic system to prevent the transmission of malaria from other parasite species. Due to the reduced chance of selective pressure against the modifications, this study also provides a novel engineered mosquito line that could be used to aid in the mitigation of malaria in high risk areas.  

As part of the validation of the novel mosquito models, Western Blot analysis to detect expression of the transgenic human PAI-1 protein was used. The authors used the Azure Imager c600 to acquire these data. In addition to chemiluminescence, the Azure c600 also detects near infrared, white light and more.

Explore: Azure c600

 

While there have been many efforts to prevent malaria transmission, research is challenging due to the complex life cycle of the parasite. However, various efforts have been made to hinder the ability of mosquitoes to carry the parasite through genetic modifications. These have shown great promise, but have not provided an ultimate solution. Usually, this genetic modification approach has focused on two main areas: suppressing or eradicating the mosquito population or modifying the mosquito population’s ability to successfully carry or transmit the parasite itself.  

While initially effective, selective pressure eventually creates a work around. To this end, Pascini et. al (2022) reported recently that they created a transgenic mosquito that did not directly target the parasite or the mosquito, but could still impair malaria transmission. This was accomplished through utilizing the mammalian fibrinolytic system, which is important for the parasitic life cycle. Because this approach does not alter the parasite directly, the risk for selective pressure is greatly reduced.

From Pascini et al. 2022 Figure 1. Immunoblotting evaluating the tissue-specific expression of PAI-1 in the transgenic mosquito. The Azure c600 was used to acquire images.  

To select a target that would affect the Plasmodium but not require changes to the parasite itself, the authors looked at outside factors involved in the life cycle of Plasmodium 

Mosquitos contract Plasmodium gametocytes through feeding on infected organisms, including humans.  Upon ingestion by the mosquito, these gametocytes travel into the midgut of the mosquito where they are fertilized and form an oocyst . The oocyst then releases thousands of sporozoites which invade the mosquito’s salivary glands.  The infected mosquito releases these sporozoites into the bloodstreams of any humans from which it takes future blood meals.  The sporozoites eventually form gametocytes. When another mosquito takes a blood meal from a newly infected human, the cycle continues.  

In both the mosquito and the human, the parasite must be able to penetrate physical barriers such as the fibrin network, extracellular matrices, and cellular barriers. Previously, Pascini and colleagues showed that some of these physical barriers were overcome by way of the mammalian fibrinolytic system.   

Fibrinolysis occurs when plasminogen is cleaved and becomes the protease plasmin, which in turn degrades fibrin, a protein found in the blood that is involved in blood clotting. This activation of plasminogen into plasmin occurs through proteolytic cleavage by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). This process is regulated by the protease inhibitor plasminogen activator inhibitor 1 (PAI-1), which inhibits tPA and uPA. 

Plasmodium makes use of this fibrinolytic system to infiltrate its host.  Both tPA and uPA are used to activate plasminogen, while plasmin helps break down the extracellular matrix, improving the parasite’s motility within the host. 

To make use of this knowledge, Pascini et al. engineered a transgenic Anopheles mosquito that constitutively expresses human PAI-1 in the midgut and/or salivary glands to target plasminogen activation on the surface of the Plasmodium parasite. While the parasite can still bind plasminogen, the ability of the parasite to activate plasminogen in either area is blocked due to the expression of PAI-1. Since the parasite makes use of plasminogen to invade and progress through its life cycle, the authors accurately predicted that presence of PAI-1 would severely impair malaria transmission.  

The authors investigated how the expression of human PAI-1 would affect the mosquitoes and found architectural changes to the salivary glands that ultimately resulted in fewer Plasmodium sporozoites in the region. Even in the midgut, they saw reduced Plasmodium infection. Reduced numbers still left the possibility for malaria transmission, as only a few sporozoites are required for infection.

The authors found expression of human PAI-1 strongly impaired transmission of malaria between hosts. This marks the first study to report a transgenic mosquito that strongly impairs Plasmodium survival through the expression of a human protein.

SOURCE
  1. Pascini, T.V., Jeong, Y.J., Huang, W. et al. Transgenic Anopheles mosquitoes expressing human PAI-1 impair malaria transmission. Nat Commun 13, 2949 (2022). https://doi.org/10.1038/s41467-022-30606-y

3 Tips for Preparing Western Blots for Publication

Categories
Imaging Western Blotting

Western blotting is a tried and true way to detect and evaluate protein expression and is widely used by researchers. While it has been around for decades, Western blots are still presented as data in both scientific talks and in published manuscripts. Getting a Western Blot that is ready for publication is the goal but as many scientists can attest, it is not always easy. The 3 tips in this blog post will help you prepare your images for publication.

There are a number of steps in the Western blot process and many of these contribute to getting Western Blot images that are publication-worthy. For instance, if you do not have adequate blocking, then you can end up with lots of background noise. While you may essentially see the answer you have been looking for, if the image of the Western blot is not ideal, it can bring into question the validity of the result all together.


1. Thoroughly Plan Your Experiment

We went into this in more detail in this article, but planning is key to having optimal Western blot images. Steps like validating the antibodies being used and ensuring all materials are not out of date can help with the overall success of the experiment. A big part of planning that affects publication is the order of your samples. Make sure you are loading the samples in a way that would be ideal for publication. For example, if you have extra lanes, you may consider adding in some additional samples to simply test if they too have a protein you will be probing for, but these samples have nothing to do with the experiment at hand. This can be a wise use of resources, but if done incorrectly, can jeopardize the chance of your Western Blot being used in a publication.

If you choose to add extra samples, add them at the end of the gel preferable with an empty lane separating them from the other samples. This allows you to easily remove them from the image for publication. If they are in the middle, then you will be forced to digitally cut them from the image, and it can raise suspicion when you have clearly pieced together the Western blot image. To avoid this altogether, be mindful of the order of samples.


2. Get a Variety of Exposures

A key part of getting publication-worthy Western blot images is the exposure and how the image is acquired. As a graduate student, I heard countless times about how I did not get the correct exposure of the Western blot. It took me a good while to figure out that it is really important to get a variety of exposures from faint to over-exposed. Having a number of exposures not only allows you to fully assess the data, but it also gives you a number of options for use in a publication.

If you’re using film, this is important because the way it looks on film may not be conveyed once you scan it so you want to have options to choose the one that best represents your data. This is one of the biggest pros of using an imager, like the Azure Sapphire. If you’re using an imager, then the imager will inherently capture a number of exposures and images for you to use from and this ultimately saves you a lot of time.

Instead of having to stand in the dark room doing a variety of exposures by hand at various lengths of time and hoping you chose the right ones, you can capture it with an imager in a matter of minutes and have a number of exposure options to choose from for your publication.

App note: Why You Should Leave the Darkroom

Explore: Azure Imaging Systems, Azure Sapphire Biomolecular Imager


3. Label and Organize

This may seem obvious, but not all scientists are naturally organized and orderly, so it warrants being said. Being organized and having all things labeled is really important. Even if you do not think the experiment is usable for a publication or a talk, you never know if it will be needed later. With this in mind, always make sure to take the time to label each lane (right on the film if you did not use an imager), add the date, and make it very clear which experiment the exposures go with. You will likely perform many Western blot experiments. It can take years to get the data needed for a paper. Instead of trying to figure out which Western blot images go with which experiment you did 2 years ago, save yourself the time and headache by making sure everything is organized and labeled well the first time.

Another tip when creating figures is to make sure you denote which Western blot image was used in the figure, including the data and exposure chosen. There are many times when you may put together a figure for lab meeting or a poster at a conference, but even if it is not for a publication, make sure to note which experiment it came from. You may end up using this figure in the manuscript after all. This will save you time and stress in the end to have these details already determined.


Conclusion

With these 3 tips, you will set yourself up to get publication-worthy Western blot images every time. Incorporating these 3 tips into your Western Blot routine will prepare you for success when it comes to time to publish your data. What are your favorite tips? Share them with us!

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How Metal Ions Impact Error-prone DNA Replication

Categories
Imaging Protein Assays

Due to the extreme specificity of genetic information, accurate DNA replication is critical to maintaining normal cell function. DNA polymerases are key proteins that catalyze DNA replication and proofread the DNA for any errors as new nucleotides are being incorporated. Without this proofreading ability, the DNA can accumulate with significant mutations. This leads to life-threatening diseases such as cancer. Therefore, the fidelity of DNA polymerases is important in DNA replication.

An example of what can occur when DNA polymerase’s proofreading capabilities are compromised is seen in individuals with mutations in the η (Pol η) gene. This polymerase is part of a family of polymerases involved in DNA translesion synthesis from UV-induced lesions. Individuals with this mutation develop hypersensitivity to UV-radiation. In some cases, an affected individual might develop skin cancer.

In recent work, Chang et. al investigated the structural components responsible for DNA polymerase fidelity. The key enzymatic reaction in DNA synthesis is the nucleotidyl transfer reaction, known to be dependent on metal ions. A variety of DNA polymerases have similar active sites containing two or three conserved acidic residues that coordinate with at least two metal ions. It was originally believed that two metal ions were sufficient for the catalysis of this reaction, but recent research has found that three metal ions are actually involved. This suggests the potential for a three-metal ion dependent process and the key structural determinants for fidelity remain unclear.

The authors made use of x-ray time-resolved crystallography to investigate the dynamic catalytic mechanism of DNA polymerase Pol η with atomic resolution. They used the metal ions Mg 2+ and Mn 2+ because each ion has been shown to impact DNA polymerase fidelity by affecting nucleotide misincorporation. In this study, Chang et al. captured the pre-, intra- and post-reaction states of Pol η misincorporating nucleotides during DNA synthesis. They discovered the accuracy of nucleotide incorporation was greatly impacted by the alignment of the primer 3’-OH

In the presence of Mn2+, DNA polymerase Pol η fidelity is lower compared to Mg2+ and Mn2+ strongly increases the efficiency of incorrect nucleotide incorporation efficiency by reducing substrate discrimination
Supplemental Figure 1 from Chang et al. (2022) In the presence of Mn2+, DNA polymerase Pol η fidelity is lower compared to Mg2+ and Mn2+ strongly increases the efficiency of incorrect nucleotide incorporation efficiency by reducing substrate discrimination. The Azure Sapphire Biomolecular Imager was used to image and quantify the gels.

The accuracy of DNA synthesis was in part determined through visualizing and quantifying DNA separation on polyacrylamide urea gels using the Azure Sapphire Biomolecular Imager. The researchers observed the third metal ion binding site had to be in an ideal position in order for nucleotidyl transfer to occur.

There was a noted difference in Mn2+ and Mg2+ with misincorporation happening more often when Mn2+ was used resulting in more error-prone polymerase catalysis of DNA synthesis.

This study highlights the essential roles of the three separate metal ions in DNA synthesis, specifically Pol η . It supports the idea that the third metal ion is catalytic and drives nucleotidyl transfer through stabilization of the transition state.

The Azure Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, near-infrared, and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

SOURCE
  1. Chang, C., Lee Luo, C. & Gao, Y. In crystallo observation of three metal ion promoted DNA polymerase misincorporation. Nat Commun 13, 2346 (2022). https://doi.org/10.1038/s41467-022-30005-3

How to Simplify Your Busy Western Blot Workflow

Categories
Western Blotting

Western blotting is a widely used technique in a variety of research fields used to detect protein in a given sample. If you are new to Western Blotting, then you are likely in the stage of learning all you can about how the procedure works and the specific steps to ensure you get accurate and valuable data. Or maybe you have been doing Western blotting for awhile, but your results are less than ideal or you find yourself repeating experiments because of different issues that come up. In this article, we are going to talk about what to consider before you do your Western blot so you do not fall into the common issues that can arise due to poor planning and preparation.

Do I really need to spend time planning my Western blot experiment?

The short answer is: yes! As many graduate students and even post docs will think, they do not have the time to plan out the experiment. There are so many other experiments to get to and this is just one of many.

Even though it seems logical to just move forward and get the data you want, the extra time to thoroughly plan and design your experiment ahead of time will prevent countless hours of frustration trying to get the Western Blot data you are needing.

Here are some compelling reasons you want to put some time into planning and preparing your Western blot experiment:

  • Limited sample – If you’ve only got a limited amount of sample or your sample is precious (i.e. patient samples or cancer tissue from a mouse model that took months to develop), then you want to ensure the Western blot is done correctly and well the first time.
  • Time – We are all busy. Planning out your experiment will save you time in the end. This could mean getting a publication out there sooner or not getting scooped. Plus, Western blots can take a long time from beginning to end. You want to set aside the time needed. Nothing is more frustrating than telling yourself you’ll be finished by 4 p.m. only to realize you don’t have enough buffer and now you have to take 30 minutes to make that.
  • Work smarter not harder – There are a number of steps involved in Western blotting and any one of those can have issues. Planning will help you avoid the pitfalls so you do not get halfway through an experiment and have to throw it out to start all over again because you realized you forgot something.
  • Data accuracy – As with every experiment, you want your data to be accurate. Planning ahead of time can help you make sure your data is both accurate and publication ready.

Ready to plan your Western blot experiment? 6 steps to follow before getting started:

 

1. Ensure you have everything you need- and that nothing is expired

While this may seem obvious, it is definitely worth the reminder. As scientists, we can get rushed and sometimes that leads to trying to cut corners. (This is especially true in those grad school and postdoc years.) But as many have learned, cutting corners does not save time in the end and instead usually causes things to take longer.

Take the extra time to make sure you have all of your materials, you have adequate amounts (do not forget to check with other lab members to make sure someone else is not planning a big experiment around the same time), and that they are not expired. Skipping this step risks your entire experiment, the sample, and the integrity of your data so it is always smart to make sure this is done. Take care of it on the front end to avoid questioning your results over an old buffer.

2. Choose the correct antibodies

While the antibody company has assured you their antibody works well for your protein when used in Western blotting, you will still want to validate the antibody yourself in your hands with your exact Western blot set up in the lab. This will give you the confidence in the results you see if you know the antibody is specific and produces little background. Or if not, then at least you know what to expect.

Another consideration when choosing primary antibodies is crossreactivity. This is another reason to do a simple test Western Blot to ensure there is not any crossreactivity. Furthermore, when choosing secondary antibodies, consider the species and any cross reactivity there.

3. Load the proper controls

It may be tempting to leave out a control to save more lanes for your samples, but choosing the necessary and proper controls for your experiment is always important. Skipping a control can require you to have to repeat the experiment again once reviewers come back with a request.  Think ahead to which controls will be needed in order to affirm the results are accurate and what a publication may require.

4. Choose the right gel percentage for your proteins of interest

This is especially important if you are looking for multiple proteins that are a similar size on the same membrane. Choose a gel percentage that will adequately separate the proteins to allow for reliable detection.

5. Plan the order of protein detection

You will likely be probing for at least 2 proteins: the protein of interest and a loading control. Often, you will want to probe for multiple proteins of interest to make your sample go as far as possible. In both cases, plan which protein you will probe for first and then subsequent proteins and their order.

6. Create a sample order that makes sense for publication

Even if this is the first Western blot for this study, set it up as though it could be used in a publication because it may. You don’t want to get to the end of the experiment to find you detected the proteins you were looking for and you have a great result, but the order of the lanes does not make sense for publication purposes. To give as many options as possible to choose from for publication, consider the order of the samples on the front end to save time.

Final Thoughts

With these 6 tips, you will be able to design your next Western Blot experiment with the best chance of avoiding these common pitfalls. Taking time to plan with these things in mind will go a long way in saving you time, money, and frustration.

COMPLETE TECHNICAL MANUAL

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices?

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Additional Resources

Document TypeDescription
App NoteWet or Dry? Which Transfer is Best for Your Assay?DOWNLOAD
App NoteHow to Improve Your Chemi BlotsDOWNLOAD
App NoteHow to Improve Your Fluorescence BlotsDOWNLOAD

Ready to learn more about how easy Western blotting can be?

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Molecular Biologist Leading the Way in Bacterium Research with the Azure c400

Categories
Customer Spotlight Imaging Western Blotting

Customer Spotlight: Tam Nguyen, PhD Candidate at Virginia Tech

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.

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.

“[We’re] investigating the host-Fusobacterium interactions and their roles in bacterial pathogenesis and altered host responses in colorectal and pancreatic cancers,” says Nguyen.

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.

Nguyen, pictured with Western blot results on the lab’s Azure c400 Imager

“We mainly use the instrument for Western blot analysis, which is routinely used in our lab for analyzing protein expression,” confirms Nguyen. “We like the chemiluminescence application because of its practicality, cost-effectiveness, and easy usage.”

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, visit their lab’s website.

Ready to learn more about how easy Western blotting can be?

Set up a free virtual demo with the Azure Imaging Systems! We'd love to meet with you and your lab.

Azure Biosystems Unveils Mini Sapphire

Categories
Imaging

Azure Biosystems Inc. is thrilled to announce the Azure Sapphire Mini, a micro, novel laser scanner that’s small but mighty. Wake up, life science, and welcome true innovation that’s going to change the way you research. Azure is proud to lead the way by pushing the boundaries of imaging as we knew it.

Modern Workflow

The Azure Sapphire Mini will allow for increased flexibility. Users will be able to add their ingredients, such as reagents and substrates, and let the Sapphire Mini do the work of putting out the perfect image. Be on the lookout for an application note in the near future using the new Sapphire Mini.

Compact, without Sacrificing the Details

With this groundbreaking scanner, we challenged ourselves to create on a different scale: the new Sapphire Mini has the same capabilities and applications as its predecessor, but in a fraction of the blueprint. Have you ever wondered what the space on your bench could be utilized for if it weren’t for the instruments occupying your real estate? The new Sapphire Mini is the smallest imaging system on the market, weighing in at only 5.33 oz.

We worked with materials experts from different industries, to come up with a solution that encapsulates the finest lasers within the new, tiny frame.

The new Sapphire Mini is ideal for delicate hands and allows for easy transfer from user to user. Plus, your clean-up will be easier than ever. Running late to an event but haven’t finished your imaging? Sapphire Mini fits into most modern pockets, and can easily connect to cellular hotspots. There’s never been a scanner quite like it.

April Fools!


While the Sapphire Mini is entirely fictional, our dedication to accelerating your science is not. We’re innovating in spaces to deliver high-performance scanning systems around the world, like the real-life Azure Sapphire™.

The Sapphire Biomolecular Imager offers fluorescent imaging in the Near IR (NIR) and Visible Wavelengths (RGB), true chemiluminescent imaging as well as scanning of storage phosphor screens. The application flexibility, sensitivity, and resolution down to 10 microns, makes it an ideal imaging system for proteomics research labs.

The Sapphire achieves high performance and application flexibility by combining up to 4 solid-state laser diodes and three different detection methods: Photo multiplier tubes (PMTs), Avalanche photodiodes (APDs) and a cooled CCD camera. Previously, labs that needed the ultimate sensitivity for chemiluminescent, phosphor imaging and fluorescent imaging, would have to buy two or three separate, high priced instruments. Combining powerful performance into one system gives researchers a choice they have never had before, fully streamlining their workflow.

Read more about the Sapphire and how it can advance your imaging game by clicking here.

Monitoring Wastewater Helps Gain Insight into the Prevalence of COVID Infection

Categories
COVID-19 qPCR

How can public health departments assess SARS-CoV-2 infection prevalence in their communities without testing and associated reporting lags?

Measuring viral COVID-19 RNA in municipal wastewater offers a way to monitor an entire community for infection load and detect undiagnosed infections, potentially providing an early indicator of local outbreaks. If accurately quantified, changes in viral RNA level can reveal trends in infection prevalence to guide public health response.

A recent report describes sensitive, robust detection of SARS-CoV-2 RNA in wastewater using the Azure Cielo 6 RT-PCR System and purification kits and the multiplex GoTaq® Enviro Wastewater SARS-CoV-2 System detection assay from Promega.

In the application note below, SARS-CoV-2 RNA levels were normalized to levels of PMMoV (a plant virus found in feces) enabling quantitative analysis of changes in SARS-CoV-2 levels over time. The resulting quantitation correlated with case counts over a 6-month period.

Documents

Document TypeDescription
Application NoteDetection of SARS-CoV-2 in WastewaterDOWNLOAD

North Carolina’s Elite Christmas Tree Industry

Categories
Customer Spotlight Imaging Western Blotting
Customer Spotlight: Adarsha Devihalli, PhD Student at NC State

 

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.

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.

“My research focuses on studying a particular strain of Phytophthora and its genetic code,” says Devihalli. “My work has initially focused on identification of the pathogen using molecular and morphological tools. However, moving forward I will be using functional genomics tools including cloning techniques. This approach will ultimately 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. 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.

Under the guidance of Dr. Whitehill, Devihalli is studying this devastating disease to better understand the issues at hand.

Two men standing in white lab coats in front of an Azure 400 imaging system on a lab bench
Adarsha and Dr. Whitehill with Azure 400 imaging system in their lab.

The process

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.

“At that point, is when the Azure 400 Imager comes in,” says Devihalli. “It’s 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.”

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].”

 

Learn about the Azure 400 Imager Devihalli uses by contacting us at info@azurebiosystems.com.
For more information on Dr. Whitehill’s Christmas tree research at NC State, visit https://research.cnr.ncsu.edu/sites/whitehilllab/

Quantitative Western Blot Quiz

Categories
Western Blotting

What’s your quantitative western blotting IQ?

Are you doing everything you should to ensure accurate quantitative Western blot data? Find out by testing yourself with this quantitative western blotting quiz. If you get one or more questions wrong, you can brush up on the basics by downloading our Quantitative Western Blotting Basics guidebook using the form on the right.

1. True or False: To get quantitative western blotting data do the following:
  • Follow your typical western blotting protocol. Be sure to probe for your protein-of-interest and a housekeeping protein so you can normalize your data
  • Image the blot on a digital imager
  • Draw boxes around the bands of your protein-of-interest and your housekeeping protein and use the imager to generate a number for band intensity. Follow your imager’s instructions for subtracting background
  • Calculate the ratio of your protein-of-interest to housekeeping protein to obtain relative protein abundance
 
2. True or False: You must use fluorescently-labeled antibodies to get quantitative western blotting data.
3. Which of the following methods can you use to validate an antibody for quantitative western blotting:

A. Genetic method: Show that when the amount of your protein-of-interest is reduced, the signal from your antibody used in an ELISA assay is also reduced.

B. Orthogonal method: Show that measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using an orthogonal method such as mass spectrometry.

C. Independent antibody: Show that the measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using a second, already validated antibody.

4. True or False: The best way to normalize western blot data is to use a housekeeping protein?
5. Which of the following parts of the western blotting workflow should be tested to ensure that your experimental conditions are not causing the signal to saturate:
SOURCE
  1. Janes KA. An analysis of critical factors for quantitative immunoblotting. Sci Signaling. 2015 Apr 7;8(371):rs2. PMCID: PMC4401487.
  2. Uhlen M, et al. A proposal for validation of antibodies. Nat Methods. 2016 Oct;13(10):823-7. PMID: 27595404.

COMPLETE TECHNICAL MANUAL

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Total Protein Normalization Stain

Categories
Western Blotting

Complete the form below to receive an email with a 25%-off promo code for your first order of AzureRed Total Protein Stain

    How to Normalize Western Blots to Total Protein

    And why normalizing to a housekeeping protein can lead you astray

    A staple of many life science labs, the western blot has evolved from the humble off-shoot of DNA and RNA blotting methods into the go-to technique for identifying specific proteins in a complex mix, verifying protein identity, and determining relative protein amounts. It’s easy, inexpensive, and the necessary instruments and reagents are widely accessible (we’re looking at you, mass spectrometry).

    One recent improvement to the technique impacts how we perform quantitative western blotting—specifically, how we normalize bands on the blot. The new recommendations to normalize to total protein instead of to a housekeeping protein should lead to western blot data that is more accurate and reproducible.

    Why Normalize to Total Protein?

    The common practice for getting quantitative/semi-quantitative data from western blots has been to normalize your band of interest to the signal from a housekeeping protein, the assumption being that being essential, the abundance of specific housekeeping proteins would be invariant across tissues and conditions. As Christian Moritz succinctly states in a 2017 Proteomics review1:

    “Over the past two decades, it became clear that this assumption is wrong.”

    Since as early as 20142, scientists have been concerned about the use of housekeeping proteins for normalizing western blots. At that time, a number of studies showed that many of the proteins commonly used for normalization, such as GAPDH, tubulin, and actin, are expressed at levels that can vary between tissue types and experimental conditions. The implications for past western blot studies is staggering, and it’s clear that moving forward researchers either need to use another normalization method to get accurate, reliable, and reproducible quantitative western blot data or else exhaustively verify that the abundance of the protein being used for normalization remains invariant across the tested conditions and is present at similar levels as the protein-of-interest.

    Another objection to using housekeeping proteins is the observation that many of them are present in much higher abundance than the protein-of-interest and, thus, are likely to be outside of the linear dynamic range of the blot1.

    Total Protein Normalization is Now the New Normal

    The emerging consensus on the best way to normalize western blots is to normalize to total protein. Many journals have embraced total protein normalization (TPN) and some, such as the Journal of Biological Chemistry, even require authors to use TPN when publishing quantitative data from western blots or else to validate the use of their housekeeping protein3, 4.

    An overview of the Total Protein Stains (TPS) and the TPN workflow

    There are a range of total protein stains (TPS) to choose from (see Mortiz1 for a nice overview of different TPS options). The TPS you choose will affect the complexity of the TPN workflow, and the important factors to consider when choosing a TPS include:

    • Dynamic range
    • Detection limits
    • Visualization method
    • Staining time
    • Visualization time
    • Consistency across tissues and experimental conditions
    • Compatibility with antibody-based detection

    Not all stains are alike and some stains are easier to use and more accurate than others. For example, there are stains which are used on the protein gel, which sounds straight-forward but can impact the efficiency of transfer to the membrane and, thus, quantitation and reproducibility. Other stains are used after transfer so will (obviously) have no impact on transfer efficiency for better accuracy and reproducibility. But if your visualization instrument is limited to two channels for detection, you will need to strip and re-probe the blot to evaluate multiple proteins, which does reduce accuracy and reproducibility.

    The TPN workflow using AzureRed Total Protein Stain

    At Azure™ Biosystems, we are dedicated to developing products that have a large positive impact on a scientist’s work while having a minimal impact on a scientist’s workflow. Our AzureRed Total Protein Stain is one example. With minimal disruption to a typical western blot workflow—there’s an additional wash/incubation step before membrane blocking—you can easily stain for (and normalize to) total protein. The process is:

    • Simple—AzureRed is a reversible stain that is compatible with downstream antibody-based detection
    • Consistent—AzureRed delivers a signal that’s reproducible and unaffected by tissue-type and experimental conditions
    • Accurate—AzureRed is linear over a wide dynamic range (> 3-log) for robust quantitation
    • Flexible—AzureRed can be used with fluorescently-labeled antibodies as well as chemiluminescent detection systems
    The TPN workflow using AzureRed Total Protein Stain

    (Left Panel) AzureRed is imaged simultaneously with three proteins of interest. Imaged on the Sapphire Biomolecular Imager. (Right Panel) AzureRed has a wider linear dynamic range compared to common housekeeping proteins.

    The workflow is simple and adds minimal time to the western blot protocol, and excitation is via the 520 nm/Cy3 channel, keeping NIR channels available for detection of multiple proteins without needing to strip and re-probe the blot.

    AzureRed Total Protein Staining Protocol

    Note that this is an abbreviated protocol. You can find the full protocol including how to use AzureRed for staining gels and how to remove AzureRed stain (AzureRed is a reversible stain) by ordering AzureRed Total Protein Stain or by downloading our Western Blotting Guidebook.

    1. Washing
    • Following transfer, wash blot for 5 min in water.
    • Proceed to PVDF (2) or Nitrocellulose (3) protocol.
    2. PVDF Protocol
    • 2a. Staining
      • Place blot protein side down in Stain Solution
      • Stain blot with gentle rocking for 15–30 min
    • 2b. Acidification
      • Place blot in Fix Solution and incubate with gentle rocking for 5 min.
    • 2c. Wash
      • Rinse blot 3 times with 100% ethanol for 2–3 min each, until green background on blot has been completely removed
    • 2d. Drying
      • Hang blot from a peg or dry on wire mesh to allow blot to dry evenly. Allow blot to dry completely before imaging

    References

    1. Mortiz CP. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics. 2017 Oct; 17(20). PMID: 28941183 (includes
      link to free full text).
    2. Ghosh R, Gilda JE, Gomes AV. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics. 2014 Oct;11(5):549-60. PMCID: PMC4791038.
    3. Collecting and presenting data. The Journal of Biological Chemistry website. http://jbcresources.asbmb.org/collecting-and-presenting-data#blot. Accessed February 4, 2019.
    4. Fosang AJ and Colbran RJ. Transparency Is the Key to Quality. J Biol Chem. Dec. 11 2015. 290(50). 29692–29694. PMCID: PMC4705984.