Western Blotting Reagents Roundup – June 2024

Categories
Reagent Roundup Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies.

Researchers trust our reagents for each step of their Western blotting workflow. Azure supports chemiluminescent and fluorescent blots with transfer membranes, protein stains, blocking and wash buffers, secondary antibodies, HRP substates, and more. Below, we’ve highlighted five recent publications that utilized Azure reagents in their Western blotting and protein analysis experiments.

Featured Studies in this Reagent Roundup

Radiance Plus HRP substrate used in Western blots as part of a study of hormone signaling in plants

Strigolactones (SL) are plant hormones regulating many aspects of plant biology including leaf development, root growth, shoot branching, and responses to pathogens, drought, and nutrient starvation. Some members of the SMXL family of proteins are rapidly degraded via a ubiquitin-like pathway in response to SL, while other members of the same family are not. Li et al1 set out to determine whether related members of this SMXL protein family can substitute for each other in Arabidopsis thaliana.

Arabidopsis thaliana leaves - multiplex image of 488nm and 520nm light sources, imaged at 10 microns with the Sapphire
Multiplex image of Arabidopsis thaliana. Imaged on the Sapphire with 488nm and 520nm laser modules at 10 µm.

Through a series of experiments, the authors found that SMXL5 (and perhaps also SMXL4) interferes with protein-protein interactions required for SL signaling, including SL-induced protein degradation. The discovery suggests that regulation of the genes encoding SMXL4 and SMXL5 could be responsible for tissue-specific differences in SL signaling. As part of this work, protein degradation was assayed by Western blot, using Radiance Plus HRP substrate, and blots were imaged on the Azure 300 imaging system.

Radiance Plus HRP substrate used in Western blots in research revealing how PTEN mutations may contribute to human neurodevelopmental diseases

Several neurodevelopmental disorders, including epilepsy and autism, are associated with mutations in the phosphatase and tensin homolog (PTEN) gene. Recent work from Dhaliwal et al2 reveals molecular mechanisms that may help explain why. Work in mice has suggested that loss of Pten results in altered signaling due to hyperactivations of mTORC1 and mTORC2. Dhaliwal et al generated human neuron, neural precursor cell, and cortical organoid experimental systems in which they could study the effects of PTEN mutations.

Western blots using Radiance plus from Azure Biosystems
Panels D, E and F from Figure 5 in Dhaliwal et al, Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids, including Western blots detected using Radiance Plus. The Western blots in panel D, quantified in panels E and F, show that double mutants of PTEN+RPTOR rescue hyperactivation of mTORC1 (pS6) and double mutants of PTEN+RICTOR rescue hyperactivation of mTORC2 (pAKT) in human neural precursor cells. Licensed under CC BY 4.0

Their results indicate that both mTORC1 and mTORC2 must be hyperactivated for PTEN mutations to cause neural changes associated with disease. Numerous Western blots were used to examine changes in protein expression in response to PTEN knockouts, visualized using Radiance Plus HRP substrate. The study suggests mTORC1 and mTORC2 could be potential therapeutic targets and that the model systems developed can serve as important tools to study human neurodevelopmental diseases.

Investigating how the stiffness of the extracellular matrix affects insulin secretion by pancreatic cells

The islets of Langerhans in the pancreas are surrounded by a unique extracellular matrix (ECM), the stiffness of which can increase in various disease states. Johansen et al3 studied the role of a mechanosensitive stretch-activated cation channel called Piezo1 in mediating changes in islet cell function and insulin secretion in response to changes in ECM stiffness. Mouse or human islet cells were encapsulated in a gel that simulated the ECM scaffold and allowed the researchers to change the stiffness of the islet cell environment.

Levels of Piezo1 protein were assessed by Western blot, using PVDF membranes, Chemi Blot Blocking Buffer, and Radiance Plus HRP substrate from Azure, and imaged on the Azure c600 imaging system. The Western blots confirmed that Piezo1 protein was present, and that its levels did not change across conditions. The study results indicate that the stiffness of the microenvironment can regulate islet cell function, and that increased stiffness impedes glucose-stimulated insulin secretion in a way that is mediated by Piezo1.

Since the release of this publication, the c600 Imaging System has been succeeded by the new Azure 600 Imaging System. This upgraded systems is a high-performance instrument capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Radiance Plus used in Western blots in a study characterizing the effects of anti-cancer agents PCAIs on breast cancer cells

Western blots using Radiance Plus
Part of Figure 4A from Lazarte et al, Activation of MAP kinase pathway by polyisoprenylated cysteinyl amide inhibitors causes apoptosis and disrupts breast cancer cell invasion, showing Western blots, detected using Radiance Plus. The Western blots were used to analyze changes in phosphorylation of MAPK pathway enzymes in cells treated with NSL-YHJ-2-27 or NSL-YHJ-2-62 at the indicated dosages for 48 h. Licensed under CC BY 4.0.

Several growth-stimulating factors that are often upregulated in cancers require KRAS protein for their action. Polyprenylated cysteinyl amide inhibitors (PCAIs) are a group of anti-cancer drugs that target KRAS and related proteins. Lazarte et al4 studied the effect of PCAIs on two breast cancer cell lines, finding that PCAIs reduced cell viability and proliferation, caused disaggregation of cell spheroids, and inhibited cell invasion into a gel. Amounts of phosphorylated proteins in the MAPK signaling pathway and of phosphorylated AKT changed in response to PCAIs.

These changes in phosphorylated protein levels were measured by Western blots detected using Radiance Plus HRP substrate. Western blotting was also used to study levels of proteins involved in apoptosis and of vinculin, a protein important for cell adhesion and migration. The results show PCAIs can disrupt multiple functions that are important for cancer cell proliferation and metastasis and suggest potential approaches to develop new therapies for some breast cancers.

AzureRed Fluorescent Protein Stain used in course of production of new class of CyTOF tags

Mass cytometry or cytometry by time-of-flight (CyTOF) is an alternative to spectral flow cytometry in the analysis of protein expression. In CyTOF, antibodies are labeled with monoisotopic metals instead of fluorescent reporters. In theory, up to 135 parameters could be detected by CyTOF in a single sample using naturally occurring elements within the appropriate mass range for the technique. In practice, only 56 have been detected and the number of available labels is limited by available chemical methods. Verhoeff et al5 created a new class of metal-complexing polymers, called HyperMAPs, that could be used to create tags for spectral CyTOF applications. The HyperMAPs consist of a peptide backbone attached to a metal-chelator complex and a biomolecule linker.

The metal-chelator complex is prepared separately and then attached to the backbone, providing greater control of metal composition, including specific ratios of different metals. After attachment of the metal-chelator complexes, the HyperMAPs can then be conjugated to antibodies via the biomolecule linker. SDS-PAGE gels stained with AzureRed Fluorescent Protein Stain were used to demonstrate that excess, unbound HyperMAP molecules were successfully removed from the completed conjugation reaction mixtures. Results of a CyTOF experiment using an antibody panel labeled with HyperMAPs were highly correlated with results using the same antibody panel labeled with traditional single isotopes.

The authors demonstrate that spectral deconvolution is possible and conclude the HyperMAPs-antibody conjugates perform at least as well as current conjugates. The new method enables 21 metals and allows greater flexibility and control of metal choice during synthesis.

SHOP: AzureRed

Browse all publications using our reagents and more on our publications list. Contact us directly for assistance with a specific product by using the form on this page.

Previous Reagent Roundups:

Read other blog posts about publications using Azure:

Shop Reagents Mentioned

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!

SOURCES

  1. Li Q, Yu H, Chang W, et al. SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation. Mol Plant. 2024;17(4):631-647.
  2. Dhaliwal N, Weng OY, Dong X, et al. Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids. Cell Rep. 2024;43(5):114173.
  3. Johansen CG, Holcomb K, Sela A, Morrall S, Park D, Farnsworth NL. Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca2+ dynamics. Matrix Biol Plus. 2024;22:100148.
  4. Lazarte JMS, Lamango NS. Activation of MAP kinase pathway by polyisoprenylated cysteinyl amide inhibitors causes apoptosis and disrupts breast cancer cell invasion. 2024;12(3):470.
  5. Verhoff J, van Asten S, Kuijper L, et al. A monodispersed metal-complexing peptide-based polymer for mass cytometry enabling spectral applications. N Biotechnol. 2024;81:33-42.

How to choose the right gel imager

Categories
Imaging

Gel imagers (also called gel docs) are typically used by research labs interested in molecular biology. An individual lab may have access to its own a gel doc, or a department may purchase a system for multiple labs to use collectively. When choosing the right gel doc, the number of parameters considered can feel overwhelming. What specific capabilities does a new imager need to have meet your research needs? Camera resolution, application choice, system size, and cost are all factors that go into making the right decision.

Modern gel imaging systems carry out many applications beyond basic documentation of gels using white or UV light. Gel imagers are light tight and if equipped with the right camera, can detect even the faintest signals. These powerful instruments can detect chemiluminescence for chemiluminescent Western blots or fluorescent signals from tissues, culture dishes, and arrays.

As image capture enthusiasts, Azure Biosystems understands better than most what goes into choosing the perfect gel imager for your lab. This article was written to guide you in choosing the best gel imager for you.

5 features to consider when choosing the right gel imager​

1. The applications you use most often

Common applications involving a gel doc include imaging DNA gels, protein gels, Western blots, and Southern blots, and colony counting on plates. Basic gel docs support fewer applications (think white-light or UV gel documentation). More advanced systems open the door to more complex applications (such as chemiluminescence, tissue imagingmultiplex fluorescence, and more).

Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Azure Imaging Systems (pictured: Azure 600 Imager) offer configurations to meet your unique combination of imaging application needs. A major perk of owning an Azure Imager is the ability to access applications such as UV, blue light, true color, trans white, near-infrared fluorescence (NIR), chemiluminescence, or visible fluorescence.

When choosing a gel imager, take inventory of the different assays you need to image. Gel imagers can carry out a variety of imaging applications to detect and analyze nucleotide or protein gels and blots. Specifications can widely vary from system to system. Each system has specific capabilities depending on how the instrument is outfitted. Do you need to image DNA gels, Western blots, or microwell plates? If you need to solely image DNA gels for genotyping purposes, the type of gel imager you need will be different than another lab that needs to be able to image gels, fluorescent membranes, and colony assays.

Additional resource: Azure Imager Comparison

2. Ease of use

Efficiency is key! When you’re busy with a long list of experiments, relying on equipment that is straightforward and easy to use saves both time and energy. Image capture software should be intuitive. When looking for a gel doc, consider how easy it will be to use it. When comparing software from system to system, choose the software that takes you from sample to saved image in the least number of steps.

We understand training users to utilize new lab equipment can be time consuming. With this in mind, the engineering team at Azure has developed an intuitive user interface that’s quick to understand, simple to operate, and effective. Our built-in software allows you to totally customize imaging protocols, while still ensuring repeatability from sample to sample.

Most customers are able to quickly operate Azure Imaging Systems without a training session. The intuitive software makes capturing images easy and reduces the time you need to spend training new lab members.

Azure Imagers also have Auto-Focus, so regardless of gel size, all images are in focus. No manual adjustments needed here.

Other ease-of-use features include Auto-Exposer, which chooses your sample’s best imaging time, and Cumulative mode, which helps you choose the best exposure for chemiluminescence samples.

3. Camera resolution and sensitivity

Compared to film, digital imaging offers wider dynamic range and more accurate quantitation. Consider the camera properties when comparing gel docs. The f-stop or aperture is an important value to consider, especially for chemiluminescence.

The quality of the data you get from a gel imager depends on the camera. The higher the megapixels, the better the resolution and clearer the images. Higher resolution images allow better differentiation of bands or spots that migrate close to each other on 1D or 2D gels. Azure Imagers use high-resolution CCD cameras, up to 9.1 MP.

Azure Imagers are designed for high sensitivity imaging and to capture maximum light output from the sample. This is achieved by having the camera directly above the sample as close as possible for best light collection efficiency (Figure 1). This direct light capture design is optimal for imaging chemiluminescence and fluorescent low light samples and consistently generates sharp, clear images.

Figure 1. Azure Imagers are designed for capturing maximum light output from the sample. The CCD sensor is placed close to the sample in order to maximize light collection efficiency which increases sensitivity. The Azure Imager camera is also directly above the sample. Having the camera as close to the sample as possible makes way for the best light collection efficiency.

Azure Imagers have a small f-stop and wide aperture. The smaller the f-stop value, the wider the aperture, and the more admitted light. Smaller f-stops drastically reduce exposure times. Lenses are now available on CCD imaging systems at f 0.95.

4. Upgradeability

A very handy (but often overlooked) feature in a gel imager is the ability to make future upgrades. There are several reasons for needing to upgrade, such as expanding research needs, initially saving budget with a lower-tier model, and more.

The Azure 200, 300, 400, and 500 can be easily upgraded to accommodate additional applications. Check out the graphic below to compare Azure Imagers.

Compare Western Blot Imaging Systems

Choose the configuration that supports today’s studies, while maintaining the option to upgrade later on

Upgrades available for chemiluminescence, NIR fluorescence and RGB fluorescence.

Not all imagers offer the option for future upgrades, so be sure to check before purchasing. Most Azure Imagers are upgradeable to include additional light sources and filter combinations. 

5. Space constraints

Gel imagers can vary significantly in size. Before purchasing a new gel imager, consider the space you have available in your lab and on your bench for the new instrument.

Systems that are operated with an adjacent external laptop or computer system can be clunky because they take up space on your bench that you might not have to spare. To save space, all Azure Imagers come with integrated touchscreens, but if you prefer to use an external PC, that option is available too.

Footprint of the Azure 600® Imager (38 cm x 55 cm x 36 cm; W x H x D) vs. the iBright® FL-1000 (dotted line; 38 cm x 60 cm x 68 cm; W x H x D)

Azure Imagers are half the size of some competing systems (Figure 2). At 42 x 56 x 33 cm, their small size translates into more bench space for other applications.

6. Bonus: Cost

The cost of gel imagers can vary from as low as $10,000 to as high as $80,000. The costs are dependent on your applications, the features you would like included in your gel doc, and add-ons. It’s always best to compare costs of similar systems across manufacturers to ensure you are getting the best system your budget allows for. If you already have an older system collecting dust, but you’re in need of a new system to better suit your needs, consider trading in your current system to offset the cost of a new Azure Imager.

Once the functionality and parameters of gel imagers are understood, you can use the information provided above to determine which gel doc is right for you. When choosing a gel doc, start by considering your research applications that require an imager. With these applications defined, the parameters needed in the gel imager are determined, and the right gel doc can be easily selected.

Considering these six aspects will allow you to select the right gel imager for you and your research needs. Once you’ve narrowed down your needs, contact an Azure rep to receive pricing on a new gel doc.

5 Latest Advancements in Cancer Research using Western Blotting and qPCR

Categories
Publication Spotlight qPCR Western Blotting

Azure Biosystems develops instrumentation to support a broad array of experimental approaches important to biomedical research.

Here we highlight five recent publications using Azure’s imaging and real-time PCR systems to advance cancer research.

1. How cancer cells evade ferroptosis

Liu et al investigated how cancer cells evade ferroptosis, a process of iron-dependent cell death that presents a promising anticancer therapeutic strategy (5). Cysteine depletion induces ferroptosis. The tumor environment is low in cysteine so cancer cells make cysteine by the reverse transsulfuration pathway which requires the enzyme cystathione b-synthase (CBS).

Chemiluminescent Western blots were imaged on the Azure c500.
Figure 4a from Liu et al, ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer showing changes in protein expression in colorectal cancer cells cultured in a cystine restricted medium for 24 hours. Chemiluminescent Western blots were imaged on the Azure c500. Licensed under CC BY 4.0.

The authors investigated how colorectal tumor cells regulate CBS. Western blots, imaged on the Azure c500 imaging system, were used to study changes in protein expression due to cysteine restriction and after knockdown of activating transcription factor 3 (ATF3). The study found ATF3 positively regulates CBS, suggesting that interfering with or blocking this signaling might enhance ferroptosis-based cancer therapy. Combined with an amino-acid restricted diet, the approach could present a new way to treat colorectal cancer.

Since the release of this publication, the c500 Imager used has been succeeded by the Azure 500 Imager, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this fluorescent imager today.

In addition to assessing miR-21 levels by qPCR, levels of PTEN and PDCD4 proteins were assessed by Western blots imaged on the Azure 600. The study found miR-21 expression to be increased in breast cancer but not correlated with age, ethnicity, or cancer subtype, suggesting it is a suitable biomarker across a variety of patient types.

2. Detecting changes in the expression of Bcl-2 in cells treated with novel nanocarriers containing anticancer agents

Tarvirdipour et al used the Sapphire Biomolecular Imager to image Western blots detecting changes in the expression of Bcl-2 in cells treated with novel nanocarriers containing anticancer agents (1). These authors developed self-assembling peptide nanocarriers designed to be taken up by cells and targeted to the nucleus while escaping endosomes without any additional modification.

Since the release of this publication, the Sapphire used has been succeeded by the Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this groundbreaking new modular imager today.

The nanocarriers were loaded with a photosensitizer and an antisense RNA nucleotide against BCL-2. The Western blot experiments demonstrated that the combined photodynamic and gene therapies reduced Bcl-2 expression to a greater extent than either therapy alone. The combined therapy also resulted in higher cytotoxicity.

3. Response of the transcriptome of neuroblastoma cells to knockdown of the DDR2 gene

Vessella et al used bulk RNA sequencing to study the response of the transcriptome of neuroblastoma cells to knockdown of the DDR2 gene when the cells were grown on collagen-coated substrates (2). The authors also investigated the effects of increasing substrate stiffness. Collagen activates DDR2, a cell surface receptor that appears to be dysregulated in many cancer types. Increased extracellular matrix (ECM) stiffness has been reported to influence cancer metastasis and the authors hypothesized DDR plays a role in cancer cells mechanosensing of their environment.

Real-time PCR on the Cielo was used to confirm the RNA-seq results; reverse-transcription qPCR reactions were performed in 96-well plates with accumulated product detected using SYBR green. The analysis surprisingly found no transcriptome changes in response to ECM stiffness. The authors propose the ECM affects cancer cells via DDR2 signaling and mechanosensing of substrate stiffness via distinct mechanisms.

4. The effect of a combination therapy on castration-resistant prostate cancer

Fischetti et al studied the effect of a combination therapy on castration-resistant prostate cancer (CRPC) (3). CPRC has poor prognosis because the cancer is resistant to androgen receptor pathway inhibitors and to immunotherapy. The authors investigated the combination of enzalutamide, an androgen-deprivation therapy (ADT)/androgen receptor pathway inhibitor (ARPI), in combination with an inhibitor of EZH2, a repressor of transcription, with the aim of restoring hormone sensitivity and improving the antitumor immune response. The combination was tested in vitro in multiple cell lines and in vivo in a mouse model.

As part of the in vitro experiments, expression of androgen receptor, EZH2, and tri-methylation of histone H3 lysine 27 were confirmed by Western blots imaged on the Azure 600 imaging system. The study results suggest that the combination therapy presents a promising avenue for future clinical investigation and may allow new immunotherapy approaches in prostate cancer.

5. Whether breast cancer expression differs with ethnicity, age, or treatment status among Malaysian women

Wong et al report the expression of miR-21 in breast cancer cases among Malaysian women, investigating whether expression differs with ethnicity, age, or treatment status (4). miR-21 is an oncogenic miRNA that is frequently upregulated in breast cancer and whose expression is associated with more advanced disease. miR-21 is proposed to act via downregulation of tumor suppressor genes PTEN and PDCD4. The majority of studies of miR-21 have involved homogeneous populations, making conclusions about relative expression levels between populations difficult. The authors aimed to study expression of the gene in the multi-ethnic Malaysian population whose access to and quality of care should be comparable.

In addition to assessing miR-21 levels by qPCR, levels of PTEN and PDCD4 proteins were assessed by Western blots imaged on the Azure 600. The study found miR-21 expression to be increased in breast cancer but not correlated with age, ethnicity, or cancer subtype, suggesting it is a suitable biomarker across a variety of patient types.

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

Set up a free virtual demo with the Azure Imaging Systems! We'd love to meet with you and your lab.
Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Revolutionizing the way you Western blot! Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence.
SOURCE
  1. Tarvirdipour S, Skowicki M, Maffeis V, Abdollahi SN, Schoenenberger C, Palivan CG. Peptide nanocarriers co-delivering an antisense oligonucleotide and photosensitizer elicit synergistic cytotoxicity. J Colloid Interface Sci. 2024;664:338-348.
  2. Vessella T, Xiang S, Xiao C, et al. DDR2 signaling and mechanosensing orchestrate neuroblastoma cell fate through different transcriptome mechanisms. FEBS Open Bio. doi: 10.1002/2211-5463.13798. Published online ahead of print.
  3. Fischetti I, Botti L, Sulsenti R, et al. Combined therapy targeting AR and EZH2 curbs castration-resistant prostate cancer enhancing anti-tumor T-cell response. 2024. doi: 10.2217/epi-2023-0374. Published online ahead of print.
  4. Wong SR, Pei CP, Islahuddin MTM, et al. miR-21 Expression and its correlation with demographics, subtypes, and tumour suppressor genes; PTEN and PDCD4 in breast cancer tissues in Malaysia. 2024;8(1):59-73.
  5. Liu J, Lu X, Zeng S, et al. ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer. Redox Biol. 2024;71:103118.

Developing a new biomanufacturing platform for adeno-associated virus

Categories
Protein Assays Publication Spotlight qPCR Western Blotting

To treat genetic diseases using gene therapy, recombinant adeno-associated viruses (AAVs) are under active investigation as vectors for gene delivery. When used for gene delivery, an AAV can achieve long-term, stable transient gene expression. An important advantage of AAVs as gene therapy vectors, compared to alternative approaches, is the ability to target gene delivery to specific tissues or cell types with cell type–specific AAV variants.

AAV therapeutics produced by new biomanufacturing platform

In recent work published in the Journal of Biological Engineering, Chen et al., describe a new biomanufacturing platform to produce AAV therapeutics that has been developed to be robust and scalable. Typically, AAV is produced in cultured cells, where the yield depends on both the amount of AAV produced by the cells and the percent of produced virus that can be recovered and purified from the culture. The authors state that current platforms are insufficient to meet demands for AAV productivity and rate of recovery. The researchers used a Cielo qPCR System and the Azure 300 Imaging System in the course of validating a new platform designed to optimize productivity and recovery (Figure 6).

characterization of AAV by silver stain (A) and chemiluminescent Western blot (B), imaged on the Azure 300
Figure 6 from Chen et al. Advanced biomanufacturing and evaluation of adeno-associated virus showing characterization of AAV by silver stain (A) and chemiluminescent Western blot (B), imaged on the Azure 300. Licensed under CC BY 4.0.

To develop the new biomanufacturing platform, the group evaluated and optimized each step of the AAV production and purification process, including selection of the host cell type, transfection parameters, culture clarification, column purification, and concentration and storage of the virus. In addition, the morphology, activity, and gene expression of the manufactured AAVs were characterized. The authors note the platform is applicable to multiple AAV serotypes.

The new platform involves producing AAV in transfected cells grown in liquid culture. The authors found VPC cells produced 5-times more AAV than HEK cells. They identified the optimal cell density and plasmid DNA to cell ratio for optimal transfection, and validated these conditions with four AAV serotypes. Fluorescent qPCR with SYBR Green detection on the Cielo was used to quantify the amount of AAV in culture. The authors assessed virus production over time, finding the optimal collection point and identified conditions to achieve 95%-100% AAV release efficiency. To scale up production, they moved production to stirred-tank bioreactors and again characterized cell growth and AAV titer over time.

Using multiple methods of column purification to identify column, buffer, and elution profiles

Producing sufficient AAV for clinical applications requires more than maximizing the amount of AAV produced by cultured cells. It is also important to extract and purify as much of the AAV as possible, while maintaining the activity of the virus. The authors characterized multiple methods of column purification to identify the combination of column, buffer, and elution profiles with the highest binding and elution rates. SDS PAGE and Western blotting were used to assess the quality of the AAV produced. Silver-stained protein gels and chemiluminescent Western blots were both imaged on the Azure 300 to detect AAV capsid proteins (see attached figure). The authors demonstrated the AAV produced in their system was active and drove functional gene expression in cell culture and in mice.

The authors conclude that the reported biomanufacturing platform is robust, scalable, and results in a high recovery rate of high-quality AAV. They emphasize that the process can be used with different AAV serotypes and could benefit numerous research and clinical applications.

Used in this study: Azure 300 Imager & Cielo qPCR System

In this study, the authors took advantage of the Azure 300 Imaging System’s ability to carry out white light (silver stain) and chemiluminescence imaging and utilized a Cielo qPCR System to quantify AAV with SYBR Green.

The Azure 300 can conduct UV and blue-light imaging and is capable of being fully upgraded to an Azure 600, a fluorescent Western blot imaging system. Learn more about the Azure 300 here.

Azure 300 imager
The Azure 300 is a multichannel, multimodal imager, with visible fluorescence, visible light, and UV excitation channels. The system is fully upgradeable to a fluorescent Western Blot Imaging System.

The Azure 300 offers the simplicity, speed and sensitivity of film detection, with better resolution and more quantitative results. This upgradeable chemiluminescent imager replaces a darkroom with film, while providing accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.

Viral titers were assessed using a Cielo. This robust qPCR system incorporates individual well scanning with channel-specific excitation and emissions fiber optics at each well for increased sensitivity and signal-to-noise. Learn more about the Cielo here.

Hundreds of clinical trials are presently studying the use of AAV-delivered gene therapies to treat numerous diseases. For any successful therapy to be produced and provided to patients, it will be necessary that the AAV therapeutic be manufactured at a large scale while maintaining activity and quality.

Gene expression as 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 Cielo qPCR system to use for one week, without any obligation, absolutely FREE.

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

Set up a free virtual demo with the Azure Imaging Systems! We'd love to meet with you and your lab.
Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Revolutionizing the way you Western blot! Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

SOURCES

  1. Chen K, Kim S, Yang S, et al. Advanced biomanufacturing and evaluation of adeno-associated virus. J Biol Eng. 2024;18(1):15.

How to Troubleshoot Common In-cell Western Issues

Categories
In-cell Western Troubleshooting

Basic guide to mastering In-Cell Western assays

In-cell Westerns (ICW) assays are powerful and versatile tools in the arsenal of cell biologists and protein research. These assays offer the unique advantage of quantifying target proteins and assessing protein expression and activation directly within the native cellular context, providing valuable insights into cellular processes, signaling pathways, and treatment responses. 

Like any experimental technique, ICWs are not immune to challenges and pitfalls. From poor antibody specificity and high background to nuances in staining, imaging platforms and quantification, these challenges can hinder progress and data interpretation.

In-cell western assay
In-cell Western assay performed using AzureCyto In-cell Western Kit. A 96-well plate was imaged on the Azure Sapphire FL using the 532, 685, and 784 Standard Optical Modules at a resolution of 10µm with the focus set to +3.00mm.

Use this basic guide to aid you in troubleshooting the common issues encountered during in-cell Western assays. Whether you are a seasoned investigator or just beginning to explore the world of ICW, this is a resource that will equip you with the knowledge and strategies you need to successfully navigate unique intricacies.

 

Common issues with In-cell Westerns

Pipette Carefully

A great rule of thumb to follow when working with cells is to never touch the bottom of the
ICW plate (or any plate for that matter) with the tip of the pipette. Touching the bottom of the plate causes defects and prevent the cells from adhering properly.

  • Pipette by touching the sides of the wells.

    To ensure the cells adhere evenly, gently tap each side of the plate a few times after seeding.

Cell Quality and Health

Poor cell health can lead to unreliable results.

  • Verify your cells are healthy, adherent, and at the appropriate confluence before initiating the assay.

  • Check for signs of contamination or cell detachment.

Determine Linear Range

For quantitative results, the number of cells plated and the concentration of primary antibody both need to be optimized prior to testing. In the case below, plating 100,000 cells per well resulted in inconsistencies since the cells were too confluent and began to peel off of the surface of the plate along the edge of the wells. Maximum linearity was observed when 1563-25,000 cells/well were used. This antibody showed good linearity at all dilutions used during optimization, so a 1:200 dilution is suitable to conserve precious and expensive antibody.

Fixation and Permeabilization

Inadequate fixation and permeabilization can result in incomplete antibody penetration and uneven staining.

  • Optimize fixation and permeabilization conditions for your specific cell type.

  • Adjust fixation and permeabilization times, concentrations, or temperatures if necessary.

  • Use a proven and robust permeabilization solution, such as AzureCyto Permeabilization solution

    The AzureCyto In-Cell Western Reagent Kit contains all reagents necessary for ICWs, including a permeabilization solution. The permeabilization solution provided with the AzureCyto Kit removes membrane lipids and facilitates permeabilization of the nuclear membrane to allow the penetration of antibodies into cells for the detection of intracellular and nuclear proteins.

  • AzureCyto In-cell Western Kit with fluorescent secondary antibodies

    AzureCyto In-Cell Western Kit

    The AzureCyto In-Cell Western Kit contains all reagents necessary for staining of whole cells for total cell normalization and simultaneous detection of two biomarkers. The …
    $749.00 (USD)

Antibody specificity

Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity

  • How to validate that your antibody can be used with the ICW technique:

    Block the antibody with a 10-fold excess of immunogen (peptide or protein) for 30 minutes prior to the antibody incubation step on the plate.

Blocking

Insufficient blocking can lead to high background signals.

  • Use an appropriate blocking buffer and incubate cells for an adequate duration.

    The block solution provided in the AzureCyto Kit is specially formulated for cell-based assays and fluorescence detection.

  • Test different blocking buffers and concentrations to minimize background.

Primary Antibody

Your choice of primary antibodies matters. Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity. Be careful: using low quality primary antibodies can yield weak or nonspecific signals.

How to select the best primaries to use when dealing with in-cell Westerns:

  • Select validated antibodies with high specificity for your target protein.

    To validate that your antibody can be used with the ICW technique, we suggest blocking the antibody with a 10-fold excess of immunogen (peptide or protein) for 30 minutes prior to the antibody incubation step on the plate.

  • Optimize antibody concentration and incubation time.

Secondary Antibody

Just like with primary antibodies, your choice of secondary antibodies matters too. If you use inappropriate or cross-reactive secondary antibodies, it can result in nonspecific staining.

Three tips for using appropriate secondary antibodies that provide quality results:

  • Choose secondary antibodies that are highly specific to the host species and isotype of your primary antibody.

  • Perform control experiments to validate secondary antibody specificity.

  • Choose fluorophores with excitation and emission ranges with little to no overlap.

    Azure offers secondary antibodies labeled with fluorophores, which emit light in visible and near-infrared wavelengths. The AzureSpectra 490, 550, 650, 700 and 800 secondary antibodies offer unparalleled sensitivity and performance for immunoblotting applications when used in conjunction with Azure Imaging Systems.

Incubation

Variations in incubation conditions can affect staining consistency. Overnight incubation times can lead to increased background.

  • Maintain consistent incubation times, temperatures, and agitation during antibody incubations.

  • Edge effects

    It is common to seed the wells of tissue culture plates with 100-200μL/well of cells in media. If the cells require incubation for more than one day prior to ICW, feed them with 50-100μL/well of fresh media. This will ensure that the cells have sufficient media for continued growth as well as prevent the wells from drying out.

common to seed the wells of tissue culture plates
Ensure there is a sufficient level of humidity inside the incubator. This can be achieved by placing a water tray filled with distilled water at the bottom of the incubator. It is typical for the wells along the edges of the plate to be impacted the most when the cells do not have sufficient media

Washing

Inadequate washing can leave residual unbound antibodies, leading to high background.

  • Ensure thorough and consistent washing between incubation steps.

  • Use an appropriate wash buffer and follow recommended wash protocols.

Imaging

Using improper imaging settings or imagers with high crosstalk can result in inaccurate data.

  • Use lasers imagers with desired fluorophores for crosstalk in the proper wavelengths.

    The Sapphire FL is capable of imaging in-cell Westerns very well, due to its sensitivity and speed. With four fluorescent channels (including two NIR channels), the Sapphire FL facilitates multiplex detection of blots and of 96-well plates with the AzureCyto In-cell Western Kit like a pro.

  • Test fluorophores and antibody pairs to determine the correct imaging settings for linearity.

  • Use consistent settings for capturing images and quantifying signals.

Normalization

Choosing the wrong normalization method can lead to misinterpretation of results.

  • Evaluate the suitability of normalization options based on your experiment.

  • Consider using total cell staining as a normalization method, especially for comparing protein levels across different samples, such as treated vs untreated cell lines.

  • Use a total stain with high linearity at the cell volume range of your experiment.

    The AzureCyto Total Cell Stain is a sensitive and linear stain that can be co-incubated with primary antibodies to streamline the detection process and reduce the number of assay steps. It is included in the AzureCyto In-cell Western Reagent Kit.

Replicates and Controls

Inadequate replicates or lack of appropriate controls can compromise data quality.

  • Include sufficient biological and technical replicates in your experiment.

  • Utilize positive and negative controls to validate assay performance.

1:2 serial dilution of HeLa cells seeded into a 96-well plate
1:2 serial dilution of HeLa cells seeded into a 96-well plate. (A) A composite image of three channels imaged simultaneously on the Sapphire™ FL at 100-micron resolution. (B) hnRNP K visualized with the 685 Standard Optical Module. (C) GAPDH visualized with the 784 Standard Optical Module. (D) AzureCyto stain visualized with the 532 Standard Optical Module. All images were taken on a Sapphire™ FL Biomolecular Scanner.

Using an in-cell Western kit such as the AzureCyto In-Cell Western Kit can cut down on the time spent troubleshooting and optimizing your assay. The AzureCyto In-Cell Western Kit enhances the consistency and reliability of results by providing researchers with validated reagents, standardized protocols, and quality controlled components reducing experimental variability and ensuring accurate and reproducible data.

Additional reading regarding in-cell Westerns

Azure to Exhibit at SFN Annual Meeting 2023 in Washington, DC

Categories
Press Releases

Dublin, Calif. – November 1, 2023 ­– Azure Biosystems, a leading innovator in life science imaging and equipment, is thrilled to announce its participation in the upcoming Society of Neuroscience 2023 Annual Meeting. The event is scheduled to take place from November 11-15, 2023 at the Walter E. Washington Convention Center, in Washington, D.C.

Visitors to the Azure booth will have the opportunity to interact with a knowledgeable team, demo the showcased equipment, and gain insights into how these state-of-the-art products can accelerate their research and discovery processes.

The Society for Neuroscience Annual Meeting is a premier event in the field of neuroscience, attracting thousands of researchers, scientists, and professionals from around the world. As a trusted name in the life sciences industry, Azure is excited to showcase its newest laser imaging system, the Sapphire FL Biomolecular Imager, its flagship imagers, the Azure Imaging Systems, as well as its reliable qPCR system, the Cielo.

Azure specializes in the development and manufacturing of state-of-the-art life science imaging solutions, catering to the specific needs of researchers and scientists in the field of neuroscience. The company’s dedication to quality, precision, and innovation has established it as a top choice for those seeking advanced chemiluminescent, fluorescent, or phosphor imaging technologies.

At the SFN 2032 Annual Meeting, Azure will be featuring its latest products and innovations, including:

  • Azure Sapphire FL Biomolecular Imager with lid open
    The Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, it easily adapts to a lab’s changing needs and advancing research. The Sapphire FL offers customizable and user-changeable optical modules, 5–1000 μm resolution scans, a Z-plane range from -1.0 to +6 mm, 5 anesthesia ports for imaging living animals, chemiluminescence detection through the Chemiluminescence Module, and much more.
  • Azure 600 Western blot Imaging system
    The Azure 600 gives you the flexibility you need for your research, while delivering solutions for quantitative Western Blot imaging. It is the only system that offers two channel, laser-based IR and chemiluminescent detection, with the speed and sensitivity of film. The Azure 600 has the ability to image visible fluorescent dyes, standard EtBr and protein gels, and excitation for quantitative Western blot imaging in the NIR. This catchall Western blot imager improves your data quality imaging with infrared dyes and offers signal stability. Low background fluorescence imaging with NIR dyes allows you to study multiple proteins in a blot, even if those proteins overlap in molecular weight.
  • Azure Cielo qPCR system
    The Azure Cielo Real-Time PCR system provides accuracy and sensitivity needed for research, with intuitive touch screen software. Its novel fiber optic detection system allows for enhanced sensitivity and speed. The Cielo is available in either 3 or 6 channels, letting you analyze a variety of commercially available dyes, already in your workflow. Onboard software operated by a 10.2″ touchscreen minimizes footprint and saves you bench space.
  • Azure chemiSOLO with lid open
    chemiSOLO is a personal chemiluminescent imager that delivers high-quality, quantitative chemiluminescent and visible protein gel imaging through a unique web-based control software. Its advanced imaging technology, intuitive controls, and compact design provide users with a personal, high-quality chemiluminescent imager that will fit neatly into their busy lab spaces. chemiSOLO provides sensitive chemiluminescent imaging with femtogram protein detection capability and up to 24-bit imaging to capture images with increased dynamic range for more quantitative Western blots.

Visit Azure at booth #802 to see how Azure can help streamline your Western blotting and imaging workflow. SFN 2023 takes place Nov. 11-15, 2023.

Is your SFN schedule quickly filling up? Schedule a meeting with someone from our team to make sure you don’t miss out! Schedule your meeting time here.

About Azure Biosystems

Azure Biosystems is a leading provider of life science imaging and equipment solutions, committed to advancing scientific research in various fields. With a focus on quality, precision, and innovation, Azure consistently develops cutting-edge products that cater to the specific needs of researchers and scientists. The company’s mission is to enable breakthrough discoveries and accelerate the pace of scientific advancement.

Contact

Tram Tran
tram.tran@azurebiosystems.com
(925) 307-7127

Common Western Blotting Questions, Answered

Categories
Fluorescence imaging Quantification Troubleshooting Western Blotting

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 (Figure 1), 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.

Answers to common Western blotting questions

Several options are available to detect Western blots, with chemiluminescence as a common option. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

ExploreWestern Blot Imaging Systems

What reagents do you need for Western bloting?

The reagents needed for Western blotting include a range of essential components, such as enhancing buffers, transfer solutions, stripping buffers, and substrates for fluorescent and chemiluminescent detection. Azure is a one-stop shop for Western blotting, offering reagents and imaging systems for detection of proteins on Western blots. Save the graphic below so you can always make sure you have the correct reagents for your next Western blot.

Western blotting steps with reagents listed
Figure 1. List of comprehensive Western blotting steps with reagents listed

Why are black dots showing on my Western blot?

If black dots are appearing on your Western blot, there may be impurities in the detection antibody you used. You can fix this by filtering the blocking reagent before using. Black dots could also mean there are aggregates in your secondary antibodies; again, filter your secondaries before use. Black dots could also appear due to aggressive stripping techniques.

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.

Depiction of chemiluminescent Western blot signal
Chemiluminescent Western blotting- one signal, one protein. In chemiluminescent detection, the antigen-primary antibody complex is bound by a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP). The enzyme catalyzes a reaction that generates light in the presence of a luminescent substrate, and the light can be detected either by exposure to x-ray film or by a CCD-based imaging system.

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

Continue readingBeginning Chemiluminescent Western Blotting

Shop chemiluminescent substratesRadiance Q

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

There are many advantages to using fluorescence to detect Western blots over chemiluminescence. The first being that fluorescent Western blotting gives you the ability to multiplex (Figure 2), which uses different fluorescent dyes with non-overlapping excitation and emission spectra, so multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.

Diagram of how fluorescent Western blotting can detect two proteins in two spectrally different channels.
Figure 2. Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

Fluorescent detection is also more quantitative than its chemiluminescent counterpart when it comes to Western blots. Because chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody, 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.

What is a chemiluminescent substrate?

Chemiluminescent substrates are made of a stable peroxide solution and an enhanced substrate solution that produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol.

An example of a chemiluminescent substrate is luminol (Figure 3), which 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.

luminol chemical formula
Figure 3. 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.

Is HRP a chemiluminescent substrate?

No! HRP is not a chemiluminescent substrate. Even though HRP is an important component of chemiluminescent detection, it 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. The location of the antibody on a blot is then detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.

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

Azure developed the 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. chemiSOLO does so without needing a designated laptop of computer- you’re able to use any smartphone or tablet.

chemiSOLO is the first imager of its kind on the market! Get a quote for chemiSOLO by clicking here or filling out the form below. We have an imager for most applications. Explore all imaging systems from Azure Biosystems.

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.

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!

Phosphor imaging with the Sapphire helps identify a keystone sRNA in Pseudomonas aeruginosa

Categories
Imaging Publication Spotlight Western Blotting

In a recent study reported in PNAS, researchers from Harvard Medical School employed the phosphor imaging capabilities of the Azure Sapphire Biomolecular imager to image a Northern blot and the quantification from a Western blot from an Azure 600 Imager to identify potential RNA targets for the many small RNAs found in the opportunistic bacteria Pseudomonas aeruginosa (P. aeruginosa)1.

Since the release of this publication, the first generation Sapphire used has been succeeded by the new Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this new imager.

Small RNAs and RNA chaperones

Small RNAs (sRNA; also known as small regulatory RNAs) are important gene expression regulators in bacteria that have the ability to respond to environmental conditions. They do not code for proteins, but serve as regulatory molecules involved in fine-tuning gene activity through binding to target mRNAs. sRNA also either inhibit translation or affect mRNA stability. They play critical roles in diverse cellular processes, including development, stress response, and immune defense 2.

RNA chaperones are specialized proteins that facilitate proper RNA folding and assist in the binding of these sRNAs and their RNA targets. They act as molecular escorts, ensuring proper structure and function of RNA molecules in various cellular contexts 3. Together, sRNAs and RNA chaperones work to precisely regulate gene expression.

Pseudomonas aeruginosa as an Opportunistic Pathogen

P. aeruginosa is a prokaryotic gram-negative, opportunistic pathogen known for its ability to thrive and adapt across diverse environments. It is responsible for over 30,000 infections among hospital patients each year and is prone to developing antibiotic resistance4. Therefore, more research on P. aeruginosa is needed to improve treatment options. The survival and adaptability of P. aeruginosa are believed to rely on changes in the expression levels of regulatory proteins, including sRNAs, to the cellular environment.

 sRNAs regulate gene expression changes at the post-transcriptional level by binding to target mRNAs and influencing their ability to be translated into protein.

P. aeruginosa harbors well over one hundred putative sRNAs. However, the precise regulatory targets of most of these sRNAs remain largely unknown. Gebhardt et al. set out to identify some of these sRNA targets and to begin to decipher the network of regulatory interactions involving sRNAs in P. aeruginosa.

Hfq: A Global Post-Transcriptional Regulator in P. aeruginosa

To begin, the researchers looked at which sRNAs bound to the prominent post-transcriptional regulator Host factor for RNA phage Qβ replication, or Hfq. Hfq is a well studied RNA chaperone that plays an important regulatory role in many different bacteria, including P. aeruginosa. Hfq facilitates the crucial base pairing interactions between sRNAs and their corresponding mRNA targets by stabilizing sRNAs and modulating their capacity to engage with target transcripts. As Hfq mediates pairing between an sRNA and a target mRNA, it can affect the translation and stability dynamics of the target mRNA species.

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.

RIL-seq: Identifying sRNA-Target Interactions in P. aeruginosa

To gain insights into the interactions between sRNAs and their targets, researchers used RNA interaction through ligation and sequencing (RIL-seq) to identify targets of Hfq-associated sRNAs. After isolating Hfq-bound RNA, RIL-seq uses computational technology to map the captured sRNA-target RNA interactions. Using this approach, interaction partners for 89 sRNAs were identified. An abundance of these RNA-RNA interactions involved one particular sRNA named PhrS.

Exploring the role of PhrS in gene regulation in P. aeruginosa

Previously, PhrS was mainly thought to function through pairing with a single target mRNA to regulate the abundance of the MvfR protein. This transcriptional regulator is required for synthesizing signals involved in quorum sensing (i.e. cell-cell communication about bacterial population densities5). Gebhardt and colleagues found PhrS binds many sRNAs, and also explored the possibility that PhrS has a more complex regulatory role that originally thought.

While MvfR affects quorum sensing in P. aeruginosa, it is not the only transcriptional regulator to do so. The transcription activator AntR negatively regulates quorum sensing by activating expression of the antABC genes.  The researchers observed that expression of two of these antABC genes  was decreased when PhrS was ectopically expressed. Since ectopic expression of an sRNA can affect the ability of native sRNAs to compete for the same targets, the researchers investigated how native levels of PhrS influence expression of the antR, antA and antB genes.

Using qRT-PCR, expression of antR, antA, and antB was measured in both wild-type and ΔphrS mutant cells . Interestingly, the results revealed that in the absence of PhrS, the expression of these genes was significantly higher, ranging from 5 to 40 times higher compared to the wild-type cells. These findings indicate that PhrS negatively regulates these genes. Whether this negative regulation depends on direct interaction between PhrS and its antABC targets remained unknown.

To begin to address whether PhrS regulates the abundance of antR mRNA through direct interaction, the team created PhrS mutants with nucleotide substitutions in the region predicted to pair with antR mRNA (the “seed” region). These substitutions were intended to disrupt normal antR-PhrSA binding.

This experiment required the use of reporters to monitor PhrS-antR mRNA interactions. Through quantification of reporter activity, the team could assess whether PhrS and antR mRNA interact directly. Expression of the antR reporter increased in ΔphrS mutant cells compared to wild-type. Remarkably, the PhrS mutants failed to repress the reporter gene, while wild-type PhrS was able to do so.  This led the researchers to conclude that PhrS represesses the translation and/or stability of the antR mRNA and that the seed region is involved in this mechanism.

Since this paper was published, the Sapphire has been succeed by the new Sapphire FL

Designed for flexible choice in detection chemistry and samples, the Sapphire FL brings precise quantitation of nucleic acids and proteins
Scientist changing optical modules on the new Azure Sapphire FL
The new Azure Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

The usage of Northern and Western blots by Gebhardt et al

While the changes in antR expression seen in the ΔphrS mutant cells do indicate regulation of the gene by PhrS, they do not definitively demonstrate direct binding of PhrS to antR mRNA. To explore this further, the researchers created 10 different phrS mutants each with dinucleotide substitutions in the predicted seed region. They then used the same reporter assay as above to examine how each mutant phrS affected antR expression. Interestingly, they discovered that mutants with mutations between positions 178 and 181 had a reduced ability to negatively regulate antR reporter expression, with the mutation of position 179 to 180 (mutant SM179) having the most profound effect.

To ensure these results were not due to varying expression of these phrS mutants, a Northern blot detected using phosphor imaging by the Sapphire was imaged (Figure 4F). Northern blots are often performed to analyze gene expression of one or a small number of genes.

Northern blotting is a technique used by molecular biologists to detect a specific RNA sequence in a sample.

Northern blotting steps >

An advantage of using this type of botting analysis over other RNA analysis techniques, is that Northern blotting visualizes the size of RNA molecules which can reveal differences in processing, such as splicing variants. The probes used on Northern blots are small RNA or DNA nucleotides. Traditionally, probes are labeled with radioisotopes, which allows for quantitation of RNA species so that expression levels can be compared between samples.

The research team observed that all but one of the PhrS mutants were as abundant as the wildtype PhrS. These data support the conclusion that the PhrS seed sequence is required for direct regulation of the antR transcript.

Northern blot used to evaluate the levels of PhrS mutants captured using Azure Sapphire Imager from Gebhardt
Figure 4F from Gehardt et al. (2023). Representative Northern blot used to evaluate the levels of each PhrS mutant. The Azure Sapphire Biomolecular Imager was used to capture images of the Northern blot shown. Licensed under CC BY 4.0.

To this point, only antR reporters had been used to investigate the PhrS-antR relationship. The researchers sought to examine whether PhrS directly pairs with antR mRNA when the gene is in its native state position in the genome. To address this question, the researchers created a mutant cell line that snythesizes AntR from its native position, but includes a VSV-G epitope tag (AntR-V) for ease of detection. They also introduced a small change (called the M2C mutation) in a specific region of the antR gene. This mutation was designed to prevent the natural PhrS from binding to the gene, but it contained the complementary bases for a specific PhrS mutant called SM179.  Therefore, this M2C mutation would allow PhrS mutant SM179 to bind to (and presumably regulate) antR.  Additionally, they created another derivative cell line that could naturally produce the PhrS mutant SM179.

Western blots imaged using the Azure 600 (Figure 4G) revealed PhrS mutant SM179 reduced the abundance of the M2C version of AntR-V, validating the direct interaction of these mutants and the regulatory nature of the interaction.

Figure 4G from Gehardt et al. (2023). Representative Western blot showing the protein levels of AntR-V with and without the restorative mutant M2C and the PhrS- SM179 mutant. The Azure 600 Biomolecular Imager was used to capture images of the Western blot. Licensed under CC BY 4.0.

How PhrS affects the abundance of quorum sensing proteins through antR mRNA regulation

Since quorum sensing signals, such as PQS, are regulated by AntR, these findings suggest that PhrS may regulate quorum sensing through direct interaction with the antR transcript.  Indeed, the researchers found that, in absence of PhrS or in the presence of M2C antR mutants (where PhrS cannot bind to antR), expression of the quorum sensing molecule PQS was reduced . These findings suggest that PhrS is involved in quorum sensing through regulation of the transcriptional regulators responsible for quorum sensing.

The Ultimate Western Blot Imaging System

The Azure 600 offers laser technology with two IR detection channels enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.
Scientist choosing settings on Azure 600
The Azure 600 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.

Gebhardt et al discovered an extensive network of RNA-RNA interactions in P. aeruginosa involving Hfq, 89 unique sRNAs, and their target transcripts. PhrS was found to be involved in the regulation of hundreds of different transcripts and affects quorum-sensing molecules through a complex mechanism that was previously unknown.

New Giveaway Announcement: Win chemiSOLO Personal Western Blot Imaging System

Categories
Press Releases

Dublin, Calif. – Sept 4, 2023 – Azure Biosystems is thrilled to announce a new giveaway campaign, giving entrants a chance to win a brand-new chemiSOLO imaging system. With this giveaway, labs from around the globe can enter to win the state-of-the-art chemiluminescent Western and visible gel imager.

The giveaway will be active from Monday, September 4, 2023 until Friday, October 20, 2023. For a chance to win a FREE chemiSOLO, click here between these dates and fill out the form on the giveaway page.

The chemiSOLO is a high-performance, personal imager that combines a compact design, unique web-based control software, and economical cost to bring high-quality imaging to any lab.
The chemiSOLO is a high-performance, personal imager that combines a compact design, unique web-based control software, and economical cost to bring high-quality imaging to any lab.

How to enter chemiSOLO giveaway

There are multiple ways to enter this giveaway. You can also increase your chance of winning by:

  • Following Azure Biosystems on Twitter
  • Following Azure Biosystems on LinkedIn
  • Signing up for the Azure Insider Newsletter
  • Tweeting a picture of where you would put your chemiSOLO in the lab using #WinchemiSOLO and tagging @azurebiosystems
Size of Azure chemiSOLO
With a footprint of only 11.5” x 17.0” x 8.75” (W x D x H), the chemiSOLO will fit neatly into any busy lab space.

“Azure Biosystems is so excited about our new chemiSOLO imaging system and we are thrilled to offer this giveaway opportunity so a lab can receive a FREE chemiSOLO,” said Toshio Kanazawa, Associate Product Manager. “This giveaway will help make state-of-the art, quantitative chemiluminescent and visible protein gel imaging accessible to more users.” 

Official rules for the chemiSOLO giveaway can be found at azurebiosystems.com/giveaway. Learn more about chemiSOLO 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 Azure Imaging Systems and the Azure Cielo Real-time PCR system. Our experienced team has applied their technical and market knowledge to develop industry standard-setting 2nd and 3rd generation imaging systems for life sciences.

Contact

Western Blotting Reagents Roundup – August 2023

Categories
Reagent Roundup Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. This quarter, we’re highlighting four recent publications that used Azure reagents to achieve excellent Western blotting results.

Featured Studies in this Reagent Roundup

AzureSpectra secondary antibodies and AzureRed Protein Stain used in a study of the effects of JAK-STAT inhibitors on thrombosis risk

Inhibition of TNF-alpha-mediated STAT1 and STAT3 with ruxolitinib and fedratinib. AzureSpectra-conjugated secondary antibodies were used to detect primary antibodies
Figure 1 from Beckman et al (2023). Inhibition of TNF-alpha-mediated STAT1 and STAT3 with ruxolitinib and fedratinib. Licensed under CC BY 4.0. AzureSpectra-conjugated secondary antibodies were used to detect primary antibodies. Western blots (Panel A) were imaged using the Azure c600 imager.

Activation of vascular endothelial cells occurs in a range of pathologic states including COVID-19 and myeloproliferative neoplasms (MPNs). The JAK-STAT signal transduction pathway is a key regulator of proinflammatory signaling. Mutations in JAK can allow ligand-independent signaling which is associated with vascular activation and increased risk of thrombosis. JAK-STAT inhibitors are being studied as potential treatments of inflammatory conditions including MPNs, COVID-19, rheumatoid arthritis, and more. However, some clinical data suggests that JAK-STAT inhibitors could increase thrombosis risk.

In recent work, Beckman et al investigated the effects of JAK-STAT inhibitors ruxolitinib and fedratinib on pro-thrombotic and pro-inflammatory signaling in endothelial cells. A variety of approaches were used to assess multiple markers of endothelial activation and cell adhesion. In one series of experiments, fluorescent Western blots were conducted to measure levels of proteins in the signaling pathway. AzureSpectra secondary antibodies labeled with visible and NIR fluorescent dyes were used for detection, and the blots were imaged on an Azure c600 imager. In addition, the blots were stained with AzureRed Protein Stain before blocking to check protein transfer. The results of the study indicate that JAK-STAT inhibitors may reduce the production of pro-inflammatory and pro-adhesive factors in endothelial cells in response to TNF-alpha stimulation.

Since the release of this publication, the c600 Imaging System has been succeeded by the new Azure 600 Imaging System. This upgraded systems is a high-performance instrument capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Study demonstrating the effectiveness of targeted pseudouridinylation to bypass premature stop codons in disease causing mutations

Several genetic diseases are caused by point mutations that change a sense codon into a stop codon. These nonsense mutations result in stop codons that cause translation to stop prematurely such that full-length proteins are not made. Premature stop codons also cause the mRNA to be degraded via nonsense-mediated mRNA decay.

In a recent publication, Adachi et al applied a strategy that they previously developed in yeast to remove the premature stop codon from a disease-causing protein in cultured human cells. Guide RNAs were used to direct the targeted conversion of the uridine in the premature stop codon into a pseudouridine. The resulting codon is no longer read as a stop codon, and the full-length protein is translated. The authors ran chemiluminescent Western blots to assess protein expression in the presence and absence of the guide RNAs. The Westerns were activated using Radiance Plus substrate and imaged on an Azure c300 imaging system. The results confirmed that targeted pseudouridylation successfully suppressed nonsense-mediated mRNA decay and promoted premature stop codon readthrough in a disease model.

Since the release of this publication, the c300 Imaging System has been succeeded by the new Azure 300 Imaging System. It offers the simplicity, speed and sensitivity of film detection, with better resolution and more quantitative results.

Total protein stain used in a study characterizing the functional consequences of a disease-causing mutation in a protein required for mitochondrial fusion

Mutations in Mfn2, a protein required for mitochondrial outer membrane fusion, cause CMT2A, Charcot-Marie-Tooth Disease Type 2, an inherited sensory motor neuropathy. 

In recent work, Sloat and Hoppins characterized the disease-causing mutation Mfn2-S350P. It is hypothesized that a large conformational change in the Hinge 2 domain of Mfn2 is important for membrane fusion. To investigate this, the authors expressed the mutant protein (and an analogous mutation in a related protein, Mfn1) in mouse cells. Abnormal clustering of mitochondria was observed. To confirm that the mitochondrial clustering was not due to altered microtubule transport, the authors knocked down expression of the dynein heavy chain protein using shRNA. Quantitative chemiluminescent Western blots activated with Radiance Plus and imaged on an Azure Sapphire Biomolecular Imager were used to confirm the knockdown.

The protein levels were quantified using a total protein stain from Azure, normalizing the signal of interest to that of total protein. The data indicate that the mutant proteins induce perinuclear clusters via mitochondrial tethering that is not dependent on dynein-mediated transport and support a model in which conformational change at the Hinge 2 domain is required to progress from tethering to membrane fusion.

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

Both Radiance and Radiance Q used in a study investigating whether the protein TSPO/PBR is required for steroidogenesis

Western blot showing VDAC-1 protein levels in various tissues visualized with Radiance ECL from Azure Biosystems.
Figure 8 from Liere et al (2023). Assessing VDAC-1 protein levels in various tissues using Western blot analysis. Licensed under CC BY 4.0. Chemiluminescence using Azure ECL Radiance or Radiance Q was used to visualize protein bands.

The protein TSPO/PBR has been thought to be required for mitochondrial cholesterol transport and therefore essential for steroid production. In their recent study, Liere et al examined the steroid profile across multiple tissues of TSPO/PBR knockout mice to determine if and how steroidogenesis depends on this protein. TSPO/PBR is highly conserved and is expressed ubiquitously, including in tissues that synthesize steroid hormones.

Prior characterization of TSPO/PBR knockout mice has focused on a small number of steroids and has not definitively answered the question of the role of TSPO/PBR in steroid synthesis.

In the present work, the authors sought to comprehensively analyze the steroid profiles of the brain, adrenal glands, testes, and plasma of male knockout mice using GC-MS/MS, a method of gas chromatography followed by mass spectrophotometry. In addition, the researchers conducted chemiluminescent Western blots to measure levels of proteins that might functionally associate with TSPO/PBR. The Westerns were activated using Azure’s Radiance and Radiance Q chemiluminescent substrates. The data revealed that TSPO/PBR has only a limited and indirect effect on steroidogenesis. The levels of proteins examined by Western blot and the levels of the majority of steroids assessed did not differ between wild type and knockout mice. The authors propose that the molecular function of TSPO/PBR requires further study.

Find more publications using Azure reagents and imaging systems on our publications list, or contact us directly for assistance with a specific product by using the form below.

Previous Reagent Roundups:

Read other blog posts about publications using Azure:

Shop Reagents Mentioned

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!

SOURCES

  1. Beckman JD, DaSilva A, Aronovich E, et al. JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions. J Thromb Haemost. 2023; S1538-7836(23)00081-8.
  2. Adachi H, Pan Y, He X, et al. Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes. Mol Cell. 2023;83:637-651.
  3. Sloat SR, Hoppins S. A dominant negative mitofusin causes mitochondrial perinuclear clusters because of aberrant tethering. Life Sci Alliance. 2023;6(1):e202101305.
  4. Liere P, Liu GJ, Pianos A, et al. The Comprehensive steroidome in complete TSPO/PBR knockout mice under basal conditions. Int J Mol Sci. 2023;24(3):2474.