Author: Tram Tran

Partnership with GenDx shows post-transplant monitoring capability using the Azure Cielo

Categories
Press Releases

Dublin, Calif. ­– November 10, 2022 – Azure Biosystems, a leading provider of innovative bioanalytical solutions for protein and genomic research, has partnered with GenDx, a leader in molecular diagnostics, to validate compatibility of the Azure Cielo 6 qPCR System with the GenDx KMRtype® and KMRtrack® kits for high-sensitivity monitoring of donor-recipient chimerism.

Azure’s work with GenDx demonstrates the Azure’s Cielo 6’s compatibility with both KMRtype and KMRtrack assays. The Cielo 6 provides the multichannel fluorescent detection required by the KMRtype assay. It can accept plate layouts from and export real-time PCR data to KMRengine, the software package from GenDx that sets up and analyzes the results of the KMRtrack and KMRtype assays. Azure and GenDx have worked together to publish an application note describing how to integrate the Cielo 6 into the associated workflows.

Read the application note using the Azure Cielo 6 with GenDx by clicking here.

After bone marrow or hematopoietic stem cell transplantation, it is important to monitor the chimeric state of the patient to ensure that donor cells survive and to quickly detect any change that would indicate a relapse of leukemia. The KMRtype and KMRtrack kits from GenDx provide a means to identify informative markers that differentiate donor from recipient cells (KMRtype) and to monitor post-transplant samples for chimerism (KMRtrack). Both kits contain the reagents and components necessary to set up multi-well plate assays that are carried out on a real-time quantitative PCR instrument.

Both the Cielo 3 and Cielo 6 are engineered for increased sensitivity and faster PCR run times compared to other RT-PCR instruments. Each LED is associated with 16 pairs of excitation and emission fibers to independently illuminate and detect 16 samples simultaneously, reducing plate movement and reducing light scatter associated with single illumination sources for excellent data consistency across different wells and different instruments.

For more information regarding Cielo or to partner on an application note, contact Azure at info@azurebiosystems.com. Download other application notes using the Azure Cielo, Azure Imaging Systems, Azure Sapphire, and more by clicking here.

About Azure Biosystems

Azure Biosystems Inc. is an innovative life science platform company that designs, develops, and markets state-of-the-art instruments, including the cSeries imaging platforms. Azure Biosystems’ experienced team has developed 2nd and 3rd generation imaging systems for the life science market, thus applying their technical and market knowledge in creating innovative industry standard setting platforms.

For additional information, please see azurebiosystems.com

About Azure Biosystems

Lisa Isailovic
lisa.isailovic@azurebiosystems.com
(925) 307-7127

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.

A malaria research study from Pascini et. al at the National Institute of Allergy and Infectious Diseases (NIAID) 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.

Western blots captured using Azure c600, published in nature
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 Western blots.  

DISCOVER: Azure 600

Research methodology

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.

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.

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.

Research surrounding malaria transmission

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.

How mosquitos contract Plasmodium

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. 

More research done with the Azure Imaging Systems:

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

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. 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.

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 shown.

Reliable data using Azure Sapphire

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.

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.

DISCOVER: Azure Sapphire Biomolecular Imager

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.

Reaction states of DNA polymerase Pol η

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. 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.

DNA polymerase, the key protein that catalyzes DNA replication

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.

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

More published research using the Azure Sapphire:

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 Western Blot Workflow

Categories
Western Blotting

Western blotting is used in a variety of research fields to used to detect how much of a protein is 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 them for awhile, but your results are less than ideal. You might be finding yourself repeating experiments because of different issues keep coming up. In this blog post, we are going to talk about what to consider before you do your next Western blot, so you do not fall into the common issues that can arise due to poor planning and preparation.

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

STEP 1: Ensure you have everything you need- and 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 (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.

Using a digital imager is another step to getting great results with your Western blots. Digital imaging provides a much larger dynamic range compared to film; low- and high-intensity bands can be imaged simultaneously. It produces a file that is immediately ready for publication, while sheets of exposed film must be  photographed or scanned to generate a digital image.

If you’re looking for an imager to image your Western blots, your search ends here. Request a free, virtual demo of an Azure Imaging System, and say “Hello” to beautiful Western blots.

Scientist standing next to Azure 600
The Azure 600 gives you the flexibility you need for your research, while delivering solutions for quantitative Western Blot imaging. Azure Biosystems provides a unified Western Blot workflow, from the high-performance imaging system and analysis software to the reagents and consumables.
Browse Western Blotting Systems

Take the extra time to make sure you have all of your materials, you have adequate amounts (check with other lab members to make sure someone else is not planning a big experiment around the same time), and your buffers are fresh, and not expired.

Skipping this step risks the sample, integrity of your data, as well your entire experiment; it is always smart to make sure your ducks are in a row. Take care of it on the front end to avoid questioning your results over something trivial, such as using an old buffer.

STEP 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. Doing a simple test Western Blot ensures there is not any crossreactivity. When choosing secondary antibodies, consider the species and any cross reactivity that might be prevalent there.

STEP 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.

STEP 4: Choose the right gel percentage for your proteins of interest

Choosing the right gel percentage is especially important if you are looking for multiple proteins that are a similar size on the same membrane. This step ensures that the gel adequately separates the proteins to allow for reliable detection.

STEP 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. Most of the time, 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.

Everything You Need to Run a Successful Western Blot

STEP 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 very well may if you play your cards right. 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.

Western Blot imaged with Azure Imager

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:

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.

The truth is, we’re 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.

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.

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.

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

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!
Download eBook

More posts on Western blotting:

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 machinery.

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!

Lemon slice - z stack image of a cross section-cut lemon, imaged with the Sapphire
Mouse embryo on Sapphire

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 COVID-19 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

The Azure Cielo makes quantitative qPCR easy as ABC

Azure is confident you will be impressed with the ease of use and performance of the Cielo has to offer. We'll arrange to send your lab a qPCR system to use for one week, without any obligation, absolutely FREE.
Start Cielo Free Trial
Azure Cielo qPCR

More qPCR blog posts:

Quantitative Western Blot Quiz

Categories
Western Blotting

True or False: To get quantitative Western blotting data do all of 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

What’s your quantitative western blotting IQ?

[qsm quiz=1]

More posts on Western blotting:

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.
QUANTITATIVE WESTERN BLOT BASICS
quantitative western blot basics

CLAIM YOUR FREE QUANTITATIVE WESTERN BLOTTING BASICS GUIDE

Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

Download Guide

Total Protein Normalization Stain

Categories
Western Blotting

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.

Overlay of four channels. Blot stained with total protein stain, AzureRed, probed for three proteins of interest without a destaining step, scanned with Azure Sapphire Biomolecular Imager
AzureRed is imaged simultaneously with three proteins of interest. The gel was loaded with dilutions of HeLa cell lysate. After transfer, the blot was stained with AzureRed and then probed for tubulin, ß-actin, and GAPDH without a destaining step. The blot was scanned with each of the four lasers of the Sapphire Biomolecular Imager. In this overlay of the four channels, total protein (AzureRed stain) is shown in gray; tubulin in red, ß-actin in blue, and GAPDH in green.

Why Should I 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. 

“Over the past two decades, it became clear that this assumption [about housekeeping proteins is essential] is wrong.”

Christian Moritz, 2017 Proteomics review

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.

AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins.
AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins.

Total protein normalization workflow using AzureRed Total Protein Stain

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. AzureRed Total Protein Stain is just one example. With minimal disruption to the 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

Delivers a signal that’s reproducible and unaffected by tissue-type and experimental conditions.

Accurate

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.

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

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

Note: 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 downloading our free Western Blotting eBook!

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!
Download eBook

Additional blog posts on total protein:

Shop AzureRed and Reagents for Normalizing to Total Protein

SOURCES

  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.

What is Western Blotting?

Categories
Fluorescence imaging Quantification Troubleshooting Western Blotting

Your common Western blotting questions, answered.

Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. It involves several steps, each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. As a leading manufacturer of Western blot imaging systems, we’re here to help. Here are some answers to your most commonly asked Western blotting questions.

What are some of the detection methods used in Western blotting?

Several options are available to detect Western blots, with chemiluminescence is likely the most common. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

Explore: Western Blot Imaging Systems

What is chemiluminescent detection?

Chemiluminescent detection is a method of detecting the location of antibodies bound to a Western blot. Chemiluminescent detection relies on an enzyme, either horseradish peroxidase or alkaline phosphatase, bound to an antibody. The enzyme converts a substrate to a product that emits light (chemiluminescence). The light emitted can be detected using a CCD camera or on X-ray film after processing in a darkroom. Developing film can be time consuming, requires access to a dedicated darkroom with appropriate equipment, and necessitates repeated purchase of reagents and single-use film. Digital imaging circumvents the development process altogether and allows labs to leave the darkroom behind. In addition to reducing the waste associated with developing film, digital imaging is more sensitive and provides a larger linear dynamic range than X-ray film. These attributes allow quantitative information to be obtained from Western blots.

What's more sensitive: chemiluminescence or fluorescence?

In general, fluorescent detection can detect picograms of protein while chemiluminescence can detect protein in the femtogram range.

However, sensitivity of detection depends on many things. The ability to detect small amounts of target protein requires a high-quality primary antibody with high affinity and specificity for the target protein. In addition, with CCD cameras, very long exposures are possible to maximize the chance of detecting a low-abundance band but this requires minimizing background “noise” on the Western blot. In addition, different fluorophores have different quantum yields, and some HRP substrates are engineered to increase sensitivity, so the sensitivity of fluorescent detection depends on the specific fluorophore used, and the sensitivity of chemiluminescent detection depends on the substrate used.

Radiance Q chemiluminescent substrate is designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data

Continue reading: Beginning Chemiluminescent Western Blotting

Explore: Radiance Q

 

What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?

Diagram of how fluorescent Western blotting can detect two proteins in two spectrally different channels.
Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

1. Fluorescent Western blotting gives you the ability to multiplex. By using different fluorescent dyes with non-overlapping excitation and emission spectra, multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.

2. Fluorescent detection is more quantitative than chemiluminescent detection. Chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody. Therefore, the activity of the enzyme can change depending on conditions and as the amount of substrate changes. Fluorescent detection relies on the emission of light from a fluorescent probe bound to the antibody. The fluorescence intensity will only depend on the number of fluorescent molecules present in a given spot.

Is HRP a chemiluminescent substrate?

Diagram illustrating the principles of chemiluminescent Western blotting
The principle of chemiluminescent Western blotting

No! Even though it is an important component of chemiluminescent detection, HRP stands for horseradish peroxidase. HRP is an enzyme that’s isolated from the roots of the horseradish plant. HRP catalyzes the oxidation of substrates, transferring electrons from the substrate to peroxide. In chemiluminescent Western blot detection, HRP is conjugated to an antibody and the location of the antibody on a blot is detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.

What is a chemiluminescence substrate?

luminol chemical formula
Luminol chemical formula. It is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected on X-ray film or by a CCD camera.

Chemiluminescent substrates produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol. Luminol is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected using a digital imager with a CCD or CMOS camera, or on X-ray film using a darkroom.

Azure chemisolo next to a hand using a mobile device to connect
A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

We developed the new chemiSOLO to make digital chemiluminescent imaging accessible to every lab. It is a personal Western blot imager that’s able to easily and quickly image chemiluminescent Western blots. It does so without needing a designated laptop of computer. With the chemiSOLO, you can image quantitative Western blots through your own mobile device or tablet. It’s the first imager of its kind on the market! Get a quote for chemiSOLO by clicking here or filling out the form below. Explore all imaging systems from Azure Biosystems by clicking here.

Additional Western Blotting Resources

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!
Download eBook

Investigating the Mechanism of Pathology in Parkinson’s Disease

Categories
Imaging

Parkinson’s disease (PD) is an age-related neurodegenerative disorder that affects almost 1 million people in the United States. PD is associated with motor symptoms including tremors and stiffness that affect balance and coordination. Symptoms appear as dopaminergic neurons are lost from the midbrain. Neuronal death appears to be due to the accumulation of toxic aggregates, called Lewy bodies, of the protein alpha-synuclein in neurons.

In recent work, Anandhan et al investigated the role of a transcription factor, NRF2, in alpha-synuclein driven pathology. NRF2 is known to regulate the cellular stress response and lack of NRF2 has been shown to exacerbate PD pathology, but the precise roll of NRF2 in alpha-synuclein driven pathology is unknown. The authors created mice that overexpress human alpha-synuclein as a model of PD. They then knocked out the gene coding for NRF2 so study the effect of the loss of NRF2 on behavioral tests, neuron loss in several brain regions, and phosphorylation and oligomerization of alpha-synuclein.

The research compared 4 groups of mice:

  1. Expressing human alpha-synuclein, expressing Nrf2 (ha-Syn+/Nrf2+)
  2. Expressing human alpha-synuclein, Nrf2 knockout (ha-Syn+/Nrf2-)
  3. Not expressing human alpha-synuclein, expressing Nrf2 (ha-Syn-/Nrf2+)
  4. Not expressing human alpha-synuclein, Nrf2 knockout (ha-Syn-/Nrf2-)
immunoblot
Figure 1. Generation of a novel humanized α-Syn/NRF2 mouse model of PD. (A) Mice overexpressing human wild-type α-Syn (hα-Syn+) were initially cross bred with Nrf2 knockout (Nrf2-/-) mice to result in hα-Syn+/Nrf2+/- and hα-Syn-/Nrf2+/- mouse strains. The hα-Syn+/Nrf2+/- mice were further crossed to finally generate four genotypes: hα-Syn+/Nrf2+/+, hα-Syn+/Nrf2-/-, hα-Syn-/Nrf2+/+ and hα-Syn-/Nrf2-/- strains. Mouse genotypes were confirmed by PCR (B and C) of tail DNA and Western blotting using Azure c600 (D).

DISCOVER: Azure 600

According to the study, lysates were boiled, sonicated, and resolved by SDS-PAGE. Membranes were subjected to appropriate antibodies at 4°C for overnight before being incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibodies from Sigma Aldrich and imaged using the Azure Biosystems c600 imager (Figure 1D).

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.

The study found increased alpha-synuclein phosphorylation and oligomerization in the mice expressing human alpha-synuclein and lacking Nrf2. These mice also lost tyrosine hydroxylase–expressing dopaminergic neurons in the substantia nigra and had behavioral defects consistent with early-state PD. The authors conclude that loss of NRF2 drives development of alpha-synuclein related PD pathogenesis via effects on oxidative stress, proteostasis, inflammation, and cell death. They suggest activating NRF2 might present a way to delay onset or progression of PD.

The Azure c600 Imager provides multiple imaging capabilities including chemiluminescence, multi-channel fluorescence, two-channel NIR fluorescence, and white light imaging of blots, gels, Petri dishes, and more. Learn more about the Azure Imaging line and how Azure can support your research by clicking here.

 

More research done with the Azure Imaging Systems: