Azure 600 helps evaluate fusion protein affinity for SARS-CoV-2 spike protein variants

Categories
COVID-19 Imaging

In a recent study from Case Western Reserve University, Matthews et al. developed a laboratory-scale production process to better evaluate fusion proteins that can neutralize emergent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants using an Azure 600 to evaluate fusion protein affinity for spike protein variants. It is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

The initial countermeasures against SARS-CoV-2 infection included vaccines, small molecule drugs, and neutralizing monoclonal antibodies. Since then, SARS-CoV-2 variants have emerged that could evade these initial countermeasures. Fusion proteins have been suggested as an ideal alternative that is not affected by variants. This work can help with fusion protein design and improving purification strategies to better study potential fusion protein treatment options.

Introduction to SARS-CoV-2, spike protein, ACE2, and treatment options

The spike (S) protein of SARS-CoV-2 infects human cells by binding to angiotensin enzyme 2 (ACE2)-expressing host cells. This leads to activation of the S protein and membrane fusion as the virus enters the cell. The ability of the S protein to bind to ACE2 is key for the virus’s pathology. The receptor binding domain (RBD) of the S protein is involved in the overall infectivity, immune evasion, and resulting transmissibility of SARS-CoV-2. Due to the high affinity of the RBD of the S protein for ACE2, a possible strategy for counteracting the virus is to use soluble ACE2 as a decoy. Ideally, ACE2 would compete for the S protein binding and thus sequester the virus to prevent it from entering cells.

More Reading: Sapphire Biomolecular Imager used in Investigation for Potential COVID-19 Nasal Vaccine

In vitro system for ACE2 fusion proteins

Previous research has that ACE2 fused with an IgG Fc region (ACE2-Fc) works best at sequestering the SARS-CoV-2 virus, but further characterization is still needed, such as determining the binding affinity for each SARS-CoV-2 variant.

Having an in vitro system to create and test ACE2-Fc proteins against SARS-CoV-2 is important to determine the best countermeasures against current and emerging variants. Matthews et. Al. (1) approached this by generating a cell line that stably expresses ACE2-Fc. The research team qualified the process for obtaining purified ACE2 fusion proteins, and created a system that allows researchers to test a hypothesis: can emerging variants that evade current countermeasures be neutralized by ACE2 fusion proteins?

Figure 1 from Matthews et al. showing upstream production of ACE2-Fc and ACE2(NN)-Fc from CHO cells. PLOS ONE 17(12): e0278294. To confirm the production of the fusion proteins from the CHO cells, immunoblots were used. Images were acquired with the Azure 600.

Creating a cell line to stably express ACE2 fusion proteins

ACE2 is part of the renin/angiotensin system, where angiotensin II regulates the cardiovascular system. ACE2 is shed into the plasma in its catalytically active form to regulate angiotensin II (2). The researchers engineered a mutant ACE2-Fc that would reduce potential effects of having an elevation of ACE2 when used as a decoy fusion protein. To abolish the enzymatic activity while retaining the binding capability to SARS-CoV-2, they mutated two residues (ACE2-(NN)-Fc).

In order to assess the binding affinity of these ACE2 fusion proteins with the S protein, researchers created a Chinese Hamster Ovary (CHO) cell line that stably expressed both ACE2-Fc and ACE2(NN)-Fc. They then confirmed the production of ACE2 fusion proteins using immunoblotting on the Azure 600 (Figure 1). Once confirmed, a laboratory-scaled protein production was performed. After purifying these recombinant proteins, Matthews et al. evaluated their stability under thermal stress, used mass spectrometry to look at their N-glycan profiles, and examined binding activity to the S protein and the different variants.

Have you published with one of our instruments? We’d love to read it! Send a link to your publication to info@azurebiosystems.com- we’ll send you something for sharing.

Characterizing the ACE2 fusion proteins

Protein stability is important for patient safety and must be maintained during the manufacturing process. Matthews et al. evaluated protein stability following different thermal-stress conditions and did not find any changes in stability at 4°C or 25°C after 1 day; however, on day 7 at 25°C, the protein was barely detectable. It is important to note the sensitivity of the ACE2-Fc proteins to thermal-induced stress discovered as this information is important for future storage and handling protocols.

Another important characterization of these fusion proteins was the binding affinity for the spike protein variants. The results found demonstrated that both ACE2-Fc and ACE2(NN)-Fc fusion proteins are active in binding to S proteins. Interestingly, they have greater affinity for alpha, beta and delta S protein variants compared to the parental and omicron variants.

Takeaways from this study

Though a clinical model for evaluating ACE2-Fc proteins as a therapeutic against SARS-CoV-2 does not currently exist, this study provides data supporting this option in the future. Matthews et al have provided data that could aid in the development of ACE2-Fc fusion proteins as a potential countermeasure against SARS-CoV-2. Furthermore, the information found can be used to support new ACE2 fusion protein designs, purification methods, and formulation studies.

The Azure 600 is one of imaging systems in the Azure Imaging Systems lineup. It improves data quality imaging with infrared dyes and offers signal stability. Low background fluorescence imaging with NIR dyes allows researchers to study multiple proteins in a blot, even if those proteins overlap in molecular weight.

To learn more about the Azure 600 and the other Azure Imaging systems, click here.

SOURCES

  1. Matthews AM, Biel TG, Ortega-Rodriguez U, Falkowski VM, Bush X, Faison T, et al. (2022) SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins. PLoS ONE 17(12): e0278294. https://doi.org/10.1371/journal.pone.0278294
  2. Turner AJ. ACE2 Cell Biology, Regulation, and Physiological Functions. The Protective Arm of the Renin Angiotensin System (RAS). 2015:185–9. doi: 10.1016/B978-0-12-801364-9.00025-0. Epub 2015 Apr 24. PMCID: PMC7149539. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149539/

Steps of Gel Electrophoresis

Categories
SDS-PAGE Western Blotting

What is gel electrophoresis?

Gel electrophoresis is a method used in the lab to separate DNA, RNA, or proteins from one another. The molecules of interest are forced through a porous gel by an electrical current, with one end of the gel being positively charged and one end being negatively charged. This results in negatively charged molecules, like DNA and RNA, traveling toward the positive end of the gel. Since proteins can have a variety of charges, they must be neutralized using sodium dodecyl sulfate (SDS) to ensure the molecule separation is not affected by charge but is only due to size. SDS also denatures proteins, preventing variations in molecule shape from affecting migration patterns.

Due to the size of the pores in the gel, larger molecules do not travel as far as smaller molecules, allowing for size separation to occur. In the end, the separated molecules can be visualized as bands1.

Tools Needed for Gel Electrophoresis

While there are different types of gel electrophoresis, the same tools are required for each type2What you’ll need to begin gel electrophoresis:

  • Gel box
  • Gel
  • Running buffer
  • A power supply
  • And visualization system.

The gel boxes differ depending on the type of gels being run. Agarose gels use horizontal gel boxes while polyacrylamide gels use vertical gel boxes, like the Azure Aqua Quad Mini Cell.

Gels are either pre-cast or hand-cast with wells at the top of the gel for the samples to be loaded into prior to their migration through the gel. The gel is submerged in running buffer containing salt ions which conduct the electrical current through the gel.

A power supply, like the Azure Aqua power supply, provides the electrical current through cables that connect to the positive and negative terminals of the gel box.

Because DNA and proteins cannot be identified with the naked eye, there must also be a way to visualize them after separation, which will be covered below.

Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside Azure Aqua Vertical Gel Running System

Steps of Gel Electrophoresis

While the specific steps of gel electrophoresis may differ somewhat between running DNA/RNA gels and protein gels, the overall steps are the same.

STEP 1: Prepare the samples

Samples will differ dramatically by individual experiment, but must all be processed similarly prior to gel electrophoresis. To begin, samples are mixed with a loading buffer. Loading buffer contains both dye, as a visual indicator while loading and running the sample, and glycerol, to increase the density of the samples. Increasing sample density promotes sinking to the bottom of the wells during loading, preventing the otherwise light samples from quickly diffusing out of the wells during loading.

STEP 2: Prepare the gel and buffer

Gels can be purchased already made (pre-cast) or they can be made in the lab (hand cast).  In preparing the gel, there are a number of factors to consider, including the gel composition, the percentage of the gel (this will affect pore size and thus separation resolution), the number of wells needed, and the size of those wells.

QUICK TIP: Buying pre-cast gels for can save time and ensure consistency of results by removing the inherent variation that comes with making gels by hand in the lab.

Choose the type of running buffer you will need and prepare it ahead of time. Buffers can often be purchased, though these are often made in bulk in the lab.

When ready to load, remove the comb from the gel. Fill the gel box with the running buffer and place the gel into the box so that it is covered by the running buffer.

STEP 3: Load samples

Before loading the samples, decide on the ideal order of the samples on the gel. Using a pipette, carefully add samples to individual wells in the gel. Additionally, a ladder with specific size markers needs to be added to one of the wells as a reference for downstream analysis.

QUICK TIP: Consider how these samples may be presented in a future figure for presentation or publication. For example, if a sample may need to be cut out of an image, it is advisable to load that sample on the end to prevent compromising the integrity of the image.

STEP 4: Electrophoresis (Running the gel)

Once the samples are loaded, place the lid on the gel box, plug the cords into the power supply, and run the gel with electrophoresis. The voltage and time required will need to be adjusted based on each lab’s specific experiment.

STEP 5: Visualize and document bands

When the steps for gel electrophoresis is complete, the resulting bands of DNA, RNA, or protein need to be visualized. For DNA gels, a DNA stain added to the gel allows visualization when placed under UV light. DNA and RNA blots require additional steps prior to visualization. Proteins can be visualized in the gel (such as with two-dimensional difference gel electrophoresis or 2D- DIGE); however, more often, they need to be transferred from the gel to a membrane for further analysis. Digital imagers, like the Azure Imagers, allow for both the visualization and documentation of results in one, swift step.

Azure chemisolo next to a hand using a mobile device to connect

Azure Biosystems offers a range of imagers capable of imaging gels stained with Coomassie, silver stain, and more. Imagers which image under white light (epi or trans-illumination) include the new chemiSOLO. This personal Western blot imager is able to easily and quickly image chemiluminescent Western blots without additional software downloads. It’s the first personal Western blot imager of its kind on the market!

Get a quote for chemiSOLO by clicking here.

The line of Azure Imaging Systems allow you to stop wasting money on film. These modular, multichannel imagers are capable of UV, color imaging, blue-excited DNA dyes, silver-stain, Coomassie gel, fluorescence imaging, and more. Azure Imagers are come equipped with visible fluorescence, visible light, and UV excitation channels and are fully upgradeable to access a wide breadth of applications.

Compare each model of the Azure Imager by clicking here.

Scientist choosing settings on Azure 600

For quick, simple confirmation of the presence of the bands, a handheld UV light or light box can also be used. 

To detect individual proteins, antibodies specific to the proteins of interest must be used. Antibodies can be designed to be detected by either chemiluminescence or fluorescence. For chemiluminescence, the protein bands can be observed using a digital imager, or with film. Fluorescence signal detection requires an imager.

Stained gels and blots can be imaged on both laser- and CCD-based fluorescent imaging systems using total protein stains, like AzureRed or Azure TotalStain Q. AzureRed is a quantitative, fluorescent protein stain for total protein normalization in blots and total protein detection in gels. It is fully compatible with downstream Western blotting or mass spectrometry. Azure TotalStain Q can be used to see all proteins on the membrane.

Different types of gel electrophoresis

Most gels used for electrophoresis are made from either agarose or polyacrylamide. DNA and RNA are separated via agarose gels while proteins are separated using polyacrylamide gels.

How long does gel electrophoresis take?

The run time for gel electrophoresis can vary anywhere from 45 to 90 minutes. The specific time needed to run a gel depends on a variety of factors, including:

  • the degree of separation desired
  • the voltage applied
  • the gel composition.

Longer run times are required for higher degrees of separation. While using a higher voltage can reduce the run time, if the voltage is too high, the gel can start to melt and create fuzzy or irregular bands. Gel composition affects how much voltage can be applied; a higher voltage may cause a low percentage gel to melt, where a higher percentage gel could withstand the higher voltage.

Troubleshooting resources for gel electrophoresis and SDS-PAGE:

SOURCES

  1. Electrophoresis. (2022, December 8). National Human Genome Research Institute. Retrieved December 15, 2022, from https://www.genome.gov/genetics-glossary/Electrophoresis
  2. DNA Gel Electrophoresis Equipment. (2019, September 11). LabXchange. Retrieved December 15, 2022, from https://www.labxchange.org/library/pathway/lx-pathway:33b08759-5d13-4128-8867-68428a8d1081/items/lx-pb:33b08759-5d13-4128-8867-68428a8d1081:html:ca030dca?source=%2Flibrary%2Fclusters%2Flx-cluster%3Aabe

Azure Imagers used to better understand the inhibitory mechanism of gut-derived colibactin production

Categories
Fluorescence imaging

The gut microbiota is made up of the trillions of microorganisms that colonize the human gut and have a significant impact on human health through their secreted products.  These microbes can be commensal or pathogenic, and some have been connected to the development of colorectal cancer. Colibactin is a common genotoxin produced in the gut by bacteria, such as E. coli, and colibactin-mediated DNA damage appears to play a role in colorectal cancer development. The Azure Sapphire and the Azure 300 imagers helped identify a small molecule inhibitor that prevents bacterial genotoxin production in a recent study by Volpe et. al1 at Harvard University.

Sapphire Helps Assess Inhibitor’s Specificity

Colibactin is a known genotoxic bacterial product produced by a non-ribosomal peptide synthetase called polyketide synthase and encoded by the gene pks. This pks gene is carried by many Escherichia coli strains (pks+ E. coli).

To determine the inhibitors’ mechanism of action, the researchers initially examined the structure of ClbP and found the inhibitors mimicked some intermediates in the hydrolysis of precolibactin.

The researchers tested the effectiveness of Inhibitor 3, the most potent of the four inhibitors, against pks+ E. coli. Using metabolomics, this inhibitor was observed to be able to block the colibactin biosynthesis while only minimally disrupting other metabolic functions.

Volpe et. al used an activity-based protein profile (ABPP) to find additional targets of their four inhibitors. In this gel-based assay, small molecules that bind to a target protein are detected by the target protein’s decreased ability to bind to a nonspecific fluorophosphonate (FP) probe compound. Using the Azure Sapphire to image the gels, no measurable differences were found (Image 4). This demonstrates a lack of additional inhibitor targets and their specificity to ClbP.

Western blots from Azure Sapphire used by Volpe et al
Image 4 from Volpe et. al. (2022) examining the efficiency of their ClbP inhibitors. The Azure Sapphire Biomolecular Imager was used in Figure 4C to examine secondary targets of the inhibitors via a ABPP assay.

Examining the Impact of Inhibitor 3 on Genotoxic Effects of Colibactin using the Azure 300

The researchers assessed whether Inhibitor 3 could inhibit the genotoxic effects of colibactin on human cells. They exposed HeLa cells to a pks+ strain of E. coli, and treated the cells with Inhibitor 3. The results indicated Inhibitor 3 is able to inhibit the genotoxic effects of colibactin in human cells, as determined by the number of cells experiencing cell-cycle arrest post treatment. It suppressed the DNA alkylating activity caused by colibactin comparable to what is observed with a genetic deletion of clbP.

Volpe et al also looked at the effects on the colibactin-induced DNA damage response. FANCD2 is a protein known to be monoubiquinated (FANCD2-Ub) in response to stalled replication forks. Previous studies show when cells are missing FANCD2, there is an increased sensitivity to colibactin3. When HeLa cells exposed to pks+ E. coli were treated with Inhibitor 3, the ubiquitination of FANCD2 was prevented, which was observed using Western blot imaged on the Azure 300.

Western blot from Volpe et al imaged with Azure 300 imager
Summary image from the abstract of Volpe et. al. representing the basic dynamic between E. coli, colibactin, and DNA damage, as well as where the created ClbP inhibitors function.

This same effect on the DNA damage response was not seen in the presence of other DNA damaging agent. This indicates Inhibitor 3 is specific to the colibactin biosynthetic pathway and not merely the DNA damage response; it ultimately inhibits the genotoxicity caused by colibactin.

Have you published with one of our instruments? We’d love to read it! Send a link to your publication to info@azurebiosystems.com- we’ll send you something for sharing.

Designing and Selecting Potential Inhibitors

Considering the proposed structure of colibactin and knowing colibactin-activating peptidase ClbP is involved in its biosynthesis, the team designed and characterized a series of inhibitors that target ClbP2.

ClbP recognizes a motif not commonly found in substrates other than colibactin. so choosing it as a target reduces the change of secondary, off-target effects from its inhibition. The researchers found four inhibitors with potential using enzymatic activity assays.

Genotoxic Bacterial Products and the Gut Microbiome

Colibactin-mediated DNA damage appears to play a role in colorectal cancer development. Additionally, colorectal cancer patients are more frequently reported to have pks+ E.coli and mouse models show an increased tumor load when colonized with pks+ E. coli. While there is a strong correlation between colibactin and colorectal cancer, markers of colibactin mutations have been observed in normal patient samples as well. This indicates the exact timing and duration of exposure to colibactin is likely an important factor influencing colorectal cancer risk and is still poorly understood.

It would be ideal to study the effects of colibactin in the natural gut environment; however, the complexity of such an environment would make it difficult to pinpoint colibactin-specific effects. Genetic manipulations of colibactin could affect the expression or functions of other genes, including structural components, and could confound the results. Since timing and exposure to colibactin is likely important in colorectal cancer development, these variables need to be accounted for in a study of this relationship.

Volpe et. al. reasoned a compound that could specifically inhibit colibactin production would allow for the complex microbiota to remain intact while assessing the specific effects of colibactin itself.

Effects of the Inhibitor on a Complex Microbial Community

Considering the inhibitors would be used in the context of the gut microbiota, the researchers examined how Inhibitor 3 would affect other members of this complex microbe community and if these conditions affected the inhibitor’s effectiveness.

These results showed in a complex microbe environment, Inhibitor 3 would likely be able to maintain its efficacy and not damage other key members of the gut microbiota.

The Findings

By targeting the biosynthetic pathway, Inhibitor 3 abrogates colibactin and completely blocks the genotoxic effects, usually observed when mammalian cells are exposed to colibactin in culture. This precise control presents an opportunity to study natural products secreted in complex microbial communities and determine potential therapeutic strategies.

Are you staying up to date on the scientific discoveries of your peers? Check out our full list of publications by visiting our Publications Database to browse publications and pre-prints using Azure Imagers and reagents. If you’re interested in speaking with an expert about any of the products Azure Biosystems provides, please send fill out the form on this page.

SOURCES

  1. Volpe, M.R., Velilla, J.A., Daniel-Ivad, M. et al. A small molecule inhibitor prevents gut bacterial genotoxin production. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01147-8
  2. Dubois, D., Baron, O., Cougnoux, A. al. ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides. Journal of Biological Chemistry (2011).
  3. Bossuet-Greif, N., Vignard, J., Taieb, F., Mirey, G., Dubois, D., Petit, C., Oswald, E., & Nougayrède, J.-P. . The colibactin genotoxin generates DNA Interstrand Cross-Links in infected cells. MBio (2018). https://doi.org/10.1128/mbio.02393-17
  4.  

Azure 300 Introduces Young Scientists to Western Blotting

Categories
Customer Spotlight Western Blotting

Customer Spotlight: Sarah Boylan, Director of The Applied Science & Engineering Program at St. Paul’s School in New Hampshire

At St. Paul’s School, a boarding school burrowed in New Hampshire, a robust and life-changing STEM program for students called The Applied Science & Engineering Program (ASEP) provides students with an opportunity to engage in real scientific research. The program’s director, Sarah Boylan, M.S., wants to ensure students have the skills needed to facilitate quality research, which means they need access to the best equipment and resources, including the Azure 300 Imager.   

The need for additional resources

When Boylan became Director of ASEP, she inherited lab equipment with the capability to do cell culture and other basic assays, like PCR. From her own experiences doing research at the Harvard Stem Cell Institute, Boylan wanted to expand the resources of the lab to broaden the research capabilities for the students. Since then, her purchases have expanded the applications ASEP students are able to run. Her initial purchases included a Nanodrop, microplate reader, and eventually, a qPCR machine. The students were able to visualize basic DNA gels, but their previous instruments were not able to image Western blots.

Chemiluminescent Western blot imaged on Azure 300 by ASEP students at St. Paul's School
Representative results generated by a student in the ASEP program using the Azure 300 Imager. Western blot reflects TcpA and Hcp Expression of V. cholerae samples grown in AKI and LB at 2hr, 4hr, and 6hr time points.

A great match with the Azure 300

Because some of the research projects included gene expression analysis, Boylan realized her students needed to have data from both qPCR and Western blots to ensure accuracy. After careful consideration, she eventually chose the Azure 300 Imager. Boylan saw the Azure 300 as the best choice for Western blot imaging at ASEP due to its affordability and ease of use. The added functionality to upgrade later on as their research capabilities expand was taken into consideration during the decision making process as well. It was clear that the Azure 300 Imager was the ideal option for ASEP.

Since acquiring the Azure 300 Imager early last year, the ASEP students have already begun implementing Western blots into their research projects. One student is examining siRNA and will use Westerns to determine the most effective concentration of siRNA to knock down a gene for cancer immunotherapy drugs. Another student is working with a cholera lab and will be using Westerns to look at two proteins involved in cholera pathogenesis.

Paving a research path at St. Paul's

These young scientists recognize that troubleshooting is an incredibly important piece of the scientific process. When gels run incorrectly or their Western blots are blank, they must learn how to identify and rectify the issues. Boylan encourages collaboration amongst the students. If one student is learning a new application, such as Western blotting or qPCR, their peers will often observe in preparation for future experiments. 

ASEP gives students the opportunity to be independent learners and thinkers, while requiring them to take full responsibility for their learning. Boylan notes that while many people may think high school students aren’t adept enough to perform some applications, she’s seen first-hand just how capable her students can be with the right resources. Some students even opt to come in during free blocks to study new material. Boylan’s goal is to ensure her students get a feel for what real research is like, including the highs and lows that come with it. Scientific research is not easy, and ASEP is designed to prepare them for that reality. Students are taught that failure is part of the process. Perseverance, commitment, and dedication will serve them in life and in their careers. 

ASEP & student life

Students begin preparing for ASEP during their junior year by securing a summer research externship at a university or company in a field of interest of their choice. After they have secured a position, they may apply to ASEP. The program is very selective and only accepts about 12 students each year. During the program, students spend the spring term of their junior year preparing for their upcoming externship.  

In the fall, students return to St. Paul’s School, where they continue their research on campus and work towards a senior capstone proposal. Some students will do an extension of their summer research and choose to work with the externship lab in an ongoing collaboration. Under Boylaa’s guidance, the young researchers determine what skills they need to ensure the best chances of success. Boylan essentially acts as a Principal Investigator overseeing twelve different research projects 

The future of science

Moving forward, Boylan hopes ASEP continues to prepare the next generation of scientists by instilling in them a love of science as well as providing a life-changing opportunity to experience real-life research. She hopes to show students and other scientists that there are more options for researchers than the rigors of academia.

Ready to expand your research horizons? Check out the capabilities of the Azure 300 Imager and discover how it can enhance your research by clicking here.

To learn more about the Applied Science and Engineering Program (ASEP) at St. Paul’s Boarding School and their research, go to https://asep.sps.edu/.

Southern blots from Sapphire confirm presence of cccDNA in Hepatitis B study

Categories
Imaging Uncategorized

Hepatitis B virus (HBV) is a deadly pathogen that affects millions worldwide every year. It can cause severe liver disease, which results in over 800,000 deaths from HBV infection every year. The treatment options available rarely cure the disease. This opens the need for new therapeutic options; however, their development is limited due to a lack of testing methods, including available animal models.

Using woodchucks as HBV models

In a recent publication published in PLOS from Pennsylvania State University College of Medicine, Hong et al tested the potential for using woodchucks (also referred to as groundhogs) as animal models for HBV. They investigated the potential cellular mechanisms responsible for species tropism by comparing woodchuck cells to human cells.

HBV has strict species and cell tropism that favors human hepatic cells, and the mechanisms behind this are not clearly understood. Researchers  are looking to develop new animal models. The first step is to understand the factors that contribute to HBV host tropism.

According to Lucifora, it is rare for current HBV therapies to see cures in chronic HBV patients because of the refractory nature of the stable closed circular DNA (cccDNA). The methods of blocking cccDNA formation and eliminating existing cccDNA pools are crucial for curing patients2.

Figure 1 from Hong et al 2022. Woodchuck cell lines were evaluated for cccDNA as an indication of HBV infection susceptibility. The Azure Sapphire Biomedical Imager was used for imaging cccDNA in Southern blots.
Figure 1 from Hong et al 2022. Woodchuck cell lines were evaluated for cccDNA as an indication of HBV infection susceptibility. The Azure Sapphire Biomedical Imager was used for imaging cccDNA in Southern blots.

Research methods using Southern blots

Hong et al looked at the ability of both hepatic and non-hepatic woodchuck cells to support HBV infection. The authors transfected cells with an HBV replicon to bypass the entry steps since HBV does not naturally infect woodchuck cells. Through a variety of assays, they found that woodchuck hepatic cell lines could indeed support HBV replication. One measure of HBV infection is to look for the presence of viral covalently cccDNA.

To confirm the presence of cccDNA, the researchers imaged Southern blots using an Azure Sapphire Biomolecular Imager. This cccDNA detection method was used throughout their research to confirm HBV infection.

The HBV entry receptor for human cells is huNTCP. After expressing huNTCP on two different woodchuck cell lines, WC3 and WCH-17, the authors exposed the cells to HBV in cell culture. Interestingly, only WCH-17 cells were able to support the HBV infection. To try and understand which factors could contribute to this difference, transcriptome analysis of the two cell lines was performed.

Since WCH-17 cells supported HBV replication when the HBV replicon was inserted into cells after the entry steps, the genes revealed with the transcriptome analysis suggests that the issue is somewhere in the process of HBV binding to the surface receptor, undergoing the initial entry steps and before nuclear import of relaxed circular DNA (rcDNA). This is common in cccDNA formation.

The research findings

Having an acceptable animal model will allow for the investigation and development of new therapeutic treatment options for those infected with HBV. This study provides new insights into the mechanisms of HBV tropism and supports the potential to develop an HBV-susceptible woodchuck model.

Background on woodchuck hepatitis virus

Woodchucks are naturally infected with woodchuck hepatitis virus (WHV). While woodchucks are not susceptible to HBV infection, WHV is similar to HBV and can be used as a model to better understand which host-specific factors affect HBV infection.

In addition to Southern blots, the Sapphire also provides a variety of other features, including multichannel fluorescence, white light imaging of tissues and gels, and fluorescent imaging of tissue culture plates. To learn more about the Sapphire and the ways Azure Biosystems can support your research, click here.

SOURCES

  1. Hong et. al. PLoS Pathog. 2022 Jun; 18(6): e1010633. Published online 2022 Jun 17. doi: 10.1371/journal.ppat.1010633

  2. Lucifora, J. & Protzer, U. Attacking hepatitis B virus cccDNA-The holy grail to hepatitis B cureJ. Hepatol. 64, S41–S48 (2016).

How SDS-PAGE Separates Proteins

Categories
SDS-PAGE

Have you ever wondered how SDS-PAGE separates proteins? As the first critical first step in the Western blotting process, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins by their molecular weight. It acts as the crucial first step to successfully detecting proteins when you are Western blotting.

What's PAGE?

PAGE is an assay by which proteins migrate through a polyacrylamide gel matrix with the application of electric current. Because of the denaturing conditions and coat of negative charge provided by SDS, the proteins will migrate based on size almost exclusively (without influence from native charge or structure). To determine the size of the sample proteins, a molecular weight marker (also referred to as a protein ladder) containing proteins of various known sizes is run alongside samples on the same gel. Often, these ladders are pre-stained so the progress of the protein can be readily visualized. Proteins can then be identified by comparing their migration patterns to those of the molecular weight marker proteins. 

How does protein travel through the gel?

The protein ladder is a purchased reagent that undergoes quality control to ensure that it is indeed prepared properly and ready for use in SDS-PAGE; however, experimental samples must be prepared in the lab. The goal of sample preparation for Western blot is denaturation and uniform coating of the proteins with a negative charge. These steps allow the proteins to travel effectively through the gel matrix based exclusively on size.

During the electrophoresis step, the proteins travel through a gel matrix, inside a gel running system (like an Azure Aqua), where an electric current pushes proteins through the gel. The current shoves the proteins to equilibrium, where they won’t move anymore.

If either of these requirements fail, protein separation will not occur properly with SDS-PAGE. The protein ladder will, of course, be unaffected by other sample preparation and will migrate properly.

Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside Azure Aqua Vertical Gel Running System

Common issues with SDS-PAGE

Unfortunately, appropriate ladder migration and separation does not guarantee the same of the sample proteins. Usually when there are migration issues with SDS-PAGE, we consider a number of factors such as buffers and gel quality. However, in this case, the successful migration and separation of the molecular weight marker indicates that those factors are working appropriately. This leads to the question of how are the protein samples differ from the protein ladder. 

Troubleshooting resources for SDS-PAGE:

Just getting started with Western blotting? This page has everything you need to prep for your first Western blot. Good luck! Use the form on this page to ask us any questions along the way. Talk to you soon in another post.

Foolproof Guide to SDS-Page Ladder Migration

Categories
SDS-PAGE Troubleshooting

You’re probably reading this post because you are experiencing ladder migration during SDS-PAGE. The good news is: we’re here to help. At Azure Biosystems, we are dedicated to helping researchers quickly obtain consistent, reliable data. Just take a look at the 3,000 publications in our resume to see for yourself. Generating reliable data depends largely on troubleshooting when things go wrong. In this blog post, I’m going to address the question:

“Why does the ladder migrate and separate as expected during SDS-PAGE, but the experimental samples do not?”

AZURE EXPERT TIP: Double-check the Sample Buffer

Proteins occur in complex tertiary and quaternary structures. For proper migration with SDS-PAGE, the proteins must be denatured so the structure does not affect their ability to migrate through the gel. To denature proteins, sample buffer should contain SDS. Additionally, the samples should be heated for further denaturation.  

In addition to the denaturation, proteins also need to be negatively charged in order to move through the gel during electrophoresis. The SDS used in the sample buffer is responsible for this step. SDS essentially coats proteins in a negative charge. 

Ensure your sample buffer contains all of the ingredients needed, in the right concentrations, and that nothing has expired. If there is an issue with migration, it is probably best to make fresh sample buffer to be safe. 

AZURE EXPERT TIP: Try heating things up

Proteins occur in complex tertiary and quaternary structures. For proper migration with SDS-PAGE, the proteins must be denatured so the structure does not affect their ability to migrate through the gel. To denature proteins, sample buffer should contain SDS. Additionally, the samples should be heated for further denaturation.  

In addition to the denaturation, proteins also need to be negatively charged in order to move through the gel during electrophoresis. The SDS used in the sample buffer is responsible for this step. SDS essentially coats proteins in a negative charge. 

Ensure your sample buffer contains all of the ingredients needed, in the right concentrations, and that nothing has expired. If there is an issue with migration, it is probably best to make fresh sample buffer to be safe.

The final step in protein denaturation is boiling the sample. While technically not required for all samples, it is good practice to include this as part of the sample preparation process. Heating adds an extra level of breaking protein bonds to linearize the proteins as much as possible.

Ensure the sample preparation protocol includes a heating step. Simply put the samples in a heat block at 95°C, for 2-5 minutes. After heating, place the samples directly on ice until ready to load into the gel. This step prevents renaturation of proteins that can occur if the samples are allowed to cool to room temperature naturally.

AZURE EXPERT TIP: Use a reducing agent

Some protein structures contain disulfide bonds. Since SDS does not disrupt disulfide bonds, a reducing agent may be added to the sample buffer to take care of this issue. The two common reducing agents used in SDS-PAGE are DTT and 𝛃-mercaptoethanol. Ensure these are added to the sample buffer to completely denature the proteins. If one is added, try making new buffer and exchanging the reducing agent used. DTT is stronger than 𝛃-mercaptoethanol so the reducing agent used could make a difference.

We hope you find these troubleshooting steps to be fairly easy to implement. With these simple changes, your bands will be separating in no time. LabXChange does a good job of explaining the purpose of proteins in SDS-PAGE using this graphic– check it out.

For more information about how Azure Biosystems can help you with analyzing proteins, check out our reagents and equipment here. If you are still experiencing issues, use the form on this page to ask our experts for help. See you next time!

Stripping and Western Blotting Part 2: Factors to Consider

Categories
Western Blotting

This is part II of our “Stripping and Western Blotting” series, so check out part I if you haven’t yet.

In the last blog, I covered the ins and outs of using stripping buffer. Now that you know which kind of stripping buffer to use and when, let’s go over when you’d want to strip and reprobe your Western blot.

One of the benefits of Western blotting is the ability to probe one membrane for multiple proteins, which gives researchers more data from one experiment, called multiplexing. Multiplexing (Figure 1) is often done by probing the same membrane for multiple targets at the same time; however, it is not always possible to probe for all targets at once. In these cases, the membrane must go through two rounds of detection. In the first round, after imaging, the membrane needs to be stripped of the original primary and secondary antibodies. During the second round, the membrane is ready to be reprobed with a different primary and secondary antibody.

Multiplex fluorescent Western blot from Azure Biosystems imager
Figure 1. Digital images of 4-color Western Blot. Using distinct fluorescent and near-infrared targeting antibodies can detect each wavelength and merge them into a four-color multiplex image. No background noise or bleeding between channels. Image captured with Azure Biosystems Sapphire Biomolecular Imager

When to strip and reprobe your Western blot

Repeating the entire Western blot process requires a lot of time, sample, and reagents. If you strip the membrane after imaging and reprobe it, you can detect additional proteins without repeating your experiment. Whether it is necessary to strip the membrane and reprobe depends on the specific experiment and situation.

Let’s look at the instances where you would strip and reprobe a Western blot membrane:

You want to look at proteins of similar molecular weight

If you are probing sequentially for proteins of a similar size, residual bands from the first protein may confound the results of the second protein. To avoid any confusion in the data, stripping the membrane removes residual primary or secondary antibodies from the first blot.

You want to avoid background noise

Even if the sample proteins are not similar in molecular weight, they could still affect the results of subsequent blots. For instance, using antibodies of the same species for proteins being detected sequentially could cause background bands to show up. During the second round of probing, using the same species-specific secondary could result in the bands from the first antibody detection round appearing.

Stripping the membrane removes the primary antibody used for detection of the first protein and prevents them from showing up in subsequent blots.  

You want to determine the relative abundance of proteins

Due to the nature of stripping, quantitative analysis is not recommended, but the relative abundance of proteins may be reliably compared.

You want to evaluate loading control proteins

Loading control proteins are used to assess relative amounts of unknown samples, which leads to the potential for higher numbers of antibody pairs when comparing loading controls to unknowns. Stripping and reprobing proves a great option in this situation.

You need to correct an error (such as using the wrong antibody)

If an accident occurs and the wrong primary antibody is added, stripping the membrane allows the experiment to be salvaged. This can save both time and resources, compared to repeating the entire experiment.

You need to fix other mistakes

A number of issues can occur throughout the Western blotting process. Some of these can be addressed by stripping and reprobing, instead of scrapping the entire experiment.

For example, if it is determined the blocking time needs to be extended, strip the antibodies, adjust the blocking time, and reprobe.

There are many steps that need optimization in the Western blotting process. Blocking reagent, primary and secondary antibody concentrations, and incubation times all contribute to the final quality of the blot. Stripping the membrane supports this optimization process by using the same membrane to determine the best conditions.

Two scientists looking at screen on Azure 600 Western blot imager

If you’re looking for a reliable imager to image your Western blots, your search ends here. Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence. Request a free, virtual demo of an Azure Imaging System, and say “Hello!” to beautiful Western blots.

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Cielo Helps Identify Potential Anti-aging Properties

Categories
qPCR

Morning glory's role on anti-aging

Plant botanicals are commonly used in skincare products, from body creams, to sunscreen and face wash. Panichakul et. al from Suan Dusit University in Thailand evaluated the anti-aging potential of Ipomoea pes-caprae (IPC), commonly known as beach morning glory or bayhops. IPC is commonly used in Thai folk medicine as a treatment for jellyfish sting-induced dermatitis.

This is the first report to show that IPC extract impacts cell proliferation, wound healing and collagen production, as well as the expression of genes involved in aging. There have also been other studies that reported on its antioxidant wound-healing properties, and found it to have components which inhibit collagenase (enzymes that break down native collagen) with little cytotoxicity. Information is constantly being discovered about the biochemical processes involved in aging. Panichakul et. al has identified compounds that may delay or even reverse the aging process at the molecular level.

Have you published with one of our instruments? We’d love to read it! Send a link to your publication to info@azurebiosystems.com- we’ll send you something for sharing.

A potential mechanism for anti-aging properties

The researchers found exposure to IPC inhibited collagenase activity. When fibroblasts were exposed to the IPC extract, there was an increase in type 1 collagen production. Beyond its structural role, type 1 collagen can also affect signaling. The signaling impacts cell proliferation and migration, which are compromised in skin aging. They found induced cell proliferation in the presence of the IPC extract.

Using a scratch assay, the researchers observed an increase in wound healing. This finding indicated that the IPC extract is capable of promoting cell migration as well.  

Panichakul et. al used the Azure Cielo to perform real-time PCR. The results from Cielo Manager Software found an increased expression of COL1A1, TGF-ꞵ1, and FGF2 in fibroblasts cells exposed to IPC extracts, the genes are essential for fibroblast proliferation, collagen production, and wound healing. The Azure Cielo is a great choice for scientists who want reliable qPCR data. Their findings from this study highlight a potential mechanism for the anti-aging properties of IPC.

In-vivo Studies using IPC extracts

Dermal fibroblasts secrete large amounts of ECM components, such as collagen. ECM components play an important role in skin aging. The researchers performed in-vitro studies using extracts of IPC on a fibroblast cell line and investigated it by evaluating its antioxidant and collagenase inhibitory activity, and ECM degradation, cell proliferation and migration.

Collagen is a major structural component of the ECM and affects the structure of the skin dermis. Degradation of collagen may lead to the increased appearance of wrinkles. The researchers reviewed collagen synthesis, which is promoted by TGF-ꞵ1 and fibroblasts growth factor 2 (FGF2).

Figure 5 from Panichakul et. al (2022) IPC extract increase expression of genes important to aspects affecting the aging of skin. Fibroblasts were exposed to IPC extract, RNA was extracted, and real-time PCR for these genes was performed using the Azure Cielo. The real-time PCR data was analyzed using the Azure Cielo Manager software.

Can extracts from IPC be used to slow down the effects of skin aging?

Results from this study suggest IPC extracts are able to reduce free radicals, inhibit collagenase, and promote cell proliferation and migration. The findings indicate IPC extracts could be used in anti-aging skin products moving forward. What do you think- will we start seeing more products on the market with IPC extracts? Is IPC the new eucalyptus or aloe vera?

What causes signs of aging?

Aging is a complex process that’s affected by intrinsic and extrinsic factors, like age, hormones, and UV exposure, that occurs in the epidermal and dermal layers of the skin. The primary cause is deterioration of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs). The breakdown of the ECM causes the skin to lose its tensile strength, which promotes the appearance of aging in the form of wrinkles, and other blemishes. Some factors, such as age, cannot be altered; however, studies on aging have revealed various prevention methods, like adding sunscreen to block damaging UV rays.

Many consumers seek new and natural therapies to combat skin wrinkling and other signs of aging. Researchers have become increasingly interested in plant extracts comprised of phytochemicals. Phytochemicals, such as flavonoids, promote cell proliferation and collagen production and are important in the aging process.

Ready to experience the Cielo for yourself? Try out the Cielo free for 7 days through our Cielo Trial Program.

More blog posts to read about qPCR:

SOURCE
  1. Panichakul T, Ponnikorn S, Tupchiangmai W, Haritakun W, Srisanga K. Skin Anti-Aging Potential of Ipomoea pes-caprae Ethanolic Extracts on Promoting Cell Proliferation and Collagen Production in Human Fibroblasts (CCD-986sk Cells). Pharmaceuticals (Basel). 2022 Aug 6;15(8):969. doi: 10.3390/ph15080969. PMID: 36015117; PMCID: PMC9416280.

Stripping and Western Blotting Part 1: How Stripping Buffer Works

Categories
Western Blotting

How many times can you strip a Western blot?

Stripping buffer works by removing the primary and secondary antibodies from the membrane. Each time you use stripping buffer, you remove a bit of the protein sample too. The number of times a membrane can be stripped and reprobed for Western blotting depends on the stripping protocol you choose and how much of the protein of interest is present. Keep in mind that eventually, the amount of the protein sample lost will be outside the minimum threshold for detection.

With that being said, if a gentle stripping protocol is applied, the process can be repeated more times than with a harsh stripping protocol. In general, a membrane can be stripped about 3 times, though some have reported stripping a membrane up to 10 times. The number of times will depend on a number of factors and can only be determined with testing.

You should always aim to set up your experiment to probe for the lowest expressed protein first. It’s easier to strip smaller amounts of antibody than larger amounts. This will be a two-part blog series. In part one, I am going to cover the main types of stripping methods. Stay tuned for part two!

The three main types of stripping methods we are going to cover in part I are: mild, harsh, and commercial. Let’s go over how long each method should take and what each entails.

Time needed: approximately 20 minutes

The mild stripping method uses a low pH glycine solution to dissociate the antibodies on the membrane from their target proteins. It does this by altering the binding site of the antibodies enough to render them inactive. 

Time needed: approximately 30-45 minutes

This method uses Tris-HCl, SDS, and a reducing agent like beta-mercaptoethanol

Time needed: If you use HRP Stripping buffer from Azure Biosystems, it only takes 5 minutes to strip the antibodies from the membrane.

Commercially available stripping buffers make the stripping process quick and easy.

Choosing the right stripping buffer

Various factors go into determining which stripping buffer is best suited for any set of conditions. For example, if the protein of interest gives a strong signal, you may want to consider starting with a harsh stripping method. If it is desirable to reuse the primary antibodies after effectively detaching them from the membrane, using a mild stripping method would be ideal.

If it is not clear which stripping buffer to choose, consider testing a mild stripping buffer first and switch to a harsher method if this is unsuccessful. 

After using a stripping buffer, test its effectiveness by probing with a secondary antibody alone and using chemiluminescent substrates to detect any signal. If the stripping process was successful, no signal should be detected. 

In the part II of Stripping and Western Blotting, I’ll discuss the scenarios where you’d want to strip and reprobe.

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SOURCES

  1. Davis, S. “Western Blot Membrane Stripping.” St John’s Laboratory, 12 November 2020, https://stjohnslabs.com/blog/western-blot-membrane-stripping. Accessed 24 September 2022.

  2. van Geldermalsen, Michelle. “Get your stripping stripes! Find out how to strip and re-blot your Western.” Bitesize Bio, 5 August 2014, https://bitesizebio.com/20884/get-your-stripping-stripes-find-out-how-to-strip-and-re-blot-your-western/. Accessed 24 September 2022.