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

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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

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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.

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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. An advantage of using Northern blotting analysis over other RNA analysis techniques is it 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.

Learn more about Northern blotting here

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

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 must the new imager have to meet your research needs? Camera resolution, application choice, system size, and cost are all factors that go into making the right decision.

A modern gel imaging system can 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), while 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, microwell plates, or more? 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. The imager’s software allows you total customization over 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; regardless of gel size, all images are in focus without needing manual adjustment. 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, capturing 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.

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.

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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.

Cielo and Sapphire aid in understanding osteoarthritis at the University of Delaware

Categories
Customer Spotlight In-cell Western Publication Spotlight qPCR

Customer Spotlight: Sofia Gonzalez, graduate student in Parreno Lab at University of Delaware (UD)

Students from the Parreno Lab at the University of Delaware (UD) use the Azure Cielo qPCR System and Sapphire Biomolecular Imager to help develop novel actin-based therapeutics to combat degenerative diseases, such as osteoarthritis.

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

Cartilage is the flexible tissue around the ends of bones at the joints that two bones to smoothly glide over one another. Osteoarthritis is one of the most common forms of arthritis and affects millions of people worldwide1. It is a degenerative joint disease that causes progressive damage to cartilage. For those affected, osteoarthritis can often lead to pain, stiffness, and ultimately loss of joint function.

Actin is a protein that plays an important role in the formation and maintenance of cartilage. When chondrocytes (the cells responsible for the maintenance of cartilage) are exposed to the cytokines associated with osteoarthritis, actin polymerization is activated2. Due to this, Principal Investigator and UD Assistant Professor Dr. Justin Parreno suspects there could be a connection between actin signaling and osteoarthritis.

Gene expression and cartilage

UD Graduate student Sofia Gonzalez is investigating the expression of an important lubricating molecule secreted by cartilage, called proteoglycan-4 (PRG-4). Gonzalez is interested in learning how to stimulate PRG-4 production; her findings could potentially prevent the onset of arthritis. However, her research path wasn’t always clear.

Initially, Gonzalez’s project began with studying hip cartilage from an ex-vivo mouse model of osteoarthritis after it was cultured for two days. She would then use the Cielo for qPCR to determine gene expression and note any changes along the way. Gene expression can tell us how a cell is functioning at a given time and is a process that allows a cell to respond to its environment. A specific protein can only be produced when its gene is turned on.

What is gene expression?

It was shortly after her initial observations that Gonzalez discovered something fascinating: the actin cytoskeleton (outer layer of cells responsible for maintaining cell structure) was changing and reorganizing itself. What she was noticing meant the gene expression changes were correlated with the cytoskeletal changes. Gonzalez has since characterized what the actin cytoskeleton looks like in chondrocytes and how it is arranged in correlation with PRG-4 expression. She has been able to emulate osteoarthritis and disease-like changes ex-vivo with mouse femoral heads. With her discovery, she seeks to evaluate PRG-4 expression in the natural environment. 

The Cielo's Popularity in the Lab

Justin Parreno and lab members using Azure Cielo Manager qPCR software
Justin Parreno and other lab members using Azure Cielo Manager qPCR software

The Parreno Lab currently has about 20 members working to understand the regulation of biological phenomena by the cytoskeleton. The students periodically use analysis of gene expression in their projects, which makes the Cielo a popular and often fully booked machine due to its ability to do semi-quantitative and quantitative PCR. Gene expression is so demanded in the Parreno lab that learning to use the Cielo is one of the first skills a student joining the lab learns. With its simple interface and reliability, Gonzalez said using it is a good way to build confidence in new students.

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.

Using the Sapphire for high-throughput screening and In-cell Westerns

In addition to using the Cielo, the Parreno lab also uses a Sapphire for a variety of projects, because they rely on the crisp and clear images it produces. The Sapphire is used to develop high-throughput screens with in-cell Westerns in 96-well plates (pictured below), with the aim of using the fluorescent intensity of proteins to determine protein regulation in chondrocytes. In-cell Westerns are useful in this case, as they allow the user to quantify proteins in cultured cells in situ to assess the effects of interventions, such as drugs, on protein levels without further manipulating the cells.

In cell western screen for Type II Collagen imaged using Azure Sapphire Biomolecular Imager
An in-cell Western scanned using the Sapphire provided by doctoral student Stephanie Richardson-Solorazno of the Parreno Lab. Eleven drugs were screened for their ability to promote cartilage deposition in cells.

In-cell Westerns quantify proteins that serve as biomarkers of disease progression. Used in research, ICWs give students and researchers the ability to assess potential therapeutics for anti-disease activity. The Sapphire is capable of in-cell westerns due to its sensitivity and speed. With four fluorescent channels including two NIR channels, the Sapphire facilitates multiplex detection like a pro.

Want to know more about in-cell Western blots? Read about them here.

Looking to the future

While the Parreno Lab has made significant progress in understanding osteoarthritis, much work remains to be done. Lab members are looking at how to quantify PRG-4 at the protein level, but she needs to develop a system to get enough protein to be able to detect it. Using mice presented the challenge of working with small bones, so the team switched to bovine cells, but are continuing to develop new assays for detecting protein in mice.

Since this interview was conducted, Gonzalez has finished her graduate degree, but her lab mates are continuing to research the biologies behind actin and cartilage and the diseases that are associated to develop novel therapeutic options for a better tomorrow.

Read more customer spotlights:

SOURCES

  1. “Osteoarthritis (OA) | Arthritis.” CDC, https://www.cdc.gov/arthritis/basics/osteoarthritis.htm. Accessed 09 February 2023
  2. “Parreno Lab.” Parreno Lab, https://parrenolab.com/research/. Accessed 11 February 2023.

How to Normalize Western Blots to Total Protein

Categories
Western Blotting

Table of Contents

Why normalizing to a housekeeping protein is not always the best method

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

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

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

Why Normalize to Total Protein

The common practice for getting quantitative/semi-quantitative data from Western blots has been to normalize your band of interest to the signal from a housekeeping protein, the assumption being that being essential, the abundance of specific housekeeping proteins would be invariant across tissues and conditions. 

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

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

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

Total Protein Normalization is the New Normal

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

Overview of Total Protein Stains (TPS) and the TPN workflow

There are a range of total protein stains to choose from (see Mortiz1 for a nice overview of different TPS options). The TPS you choose will affect the complexity of the TPN workflow.

Important factors to consider when choosing a TPS:

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

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

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

Total protein normalization workflow using AzureRed Total Protein Stain

We are dedicated to developing products that have a large positive impact on your work while having a minimal impact on your current workflow. AzureRed Total Protein Stain is just one example. With minimal disruption to the typical Western blot workflow—there’s an additional wash/incubation step before membrane blocking—you can easily stain for (and normalize to) total protein. Using AzureRed is simple, consistent, accurate, and flexible. More benefits to using it are listed in Table 1.

Table 1. Perks to incorporating AzureRed into your current Western blot workflow

AzureRed is...
Simple to useReversible and compatible with downstream antibody-based detection
ConsistentDelivers a signal that’s reproducible and unaffected by tissue-type and experimental conditions. It is designed to preserve a consistent pH level, aiding the elution of proteins from the gel, as well as their binding to the membrane
AccurateLinear over a wide dynamic range (> 3-log) for robust quantitation
FlexibleCan be used with fluorescently-labeled antibodies, as well as the Azure Imagers or other chemiluminescent detection systems

The AzureRed workflow (Figure 2) is simple and adds minimal time to the Western blot protocol. Excitation is via the 520 nm/Cy3 channel on the Sapphire Imager.

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

(Left Panel) Image of a fluorescent multiplex Western blot with three proteins of interest using AzureRed. Imaged on the Azure Sapphire Biomolecular Imager. (Right Panel) AzureRed total protein stain has a wider linear dynamic range compared to common housekeeping proteins.
Figure 2. TPN workflow using AzureRed Total Protein Stain. (Left Panel) AzureRed is imaged simultaneously with three proteins of interest. Imaged on the Sapphire Biomolecular Imager. (Right Panel) AzureRed has a wider linear dynamic range compared to common housekeeping proteins.

Using the Cy3 channel keeps the NIR channels available for detection of multiple proteins, without needing to strip and re-probe the Western blot. You can also remove AzureRed after staining as it is a removeable stain.

AzureRed Total Protein Staining Short Protocol

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

Note: The above is an abbreviated protocol. You can find the full protocol including how to use AzureRed for staining gels and how to remove it afterwards by downloading our free Western Blotting eBook to learn the best tips and tricks for Western blotting.

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!

Additional blog posts on total protein:

Shop AzureRed and Reagents for Normalizing to Total Protein

SOURCES

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

Characterizing candidate anticancer drugs through fluorescent imaging with the Azure c600

Categories
Fluorescence imaging Protein Assays Quantification

Cisplatin is a chemotherapeutic agent used to treat many types of solid tumors, including hematologic cancers. Its anti-tumor effects result from platinum binding covalently to purine bases, leading to DNA damage and cell death. Because cisplatin and other platinum agents are associated with toxicity, researchers are searching for replacements to conventional platinum chemotherapy. In a recent publication from Mendel University in Zemedelska, Mitrevska et al used the Azure c600 as part of a study to characterize candidate anticancer drugs and compare the anticancer activity of platinum nanoparticles to cisplatin using a breast cancer model.

Since the release of this publication, the cSeries Imaging Systems have been succeeded by the new Azure Imaging Systems. The upgraded systems are high performance instruments capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Using in ovo and ex ovo chick CAM assays

Chick chorioallantoic membrane (CAM) assays are used to study angiogenesis and tumor biology, and are faster and less expensive to use than mouse models. They provide an in vivo system to study tumor vascularization and metastasis. To implant the tumor cells into the chorioallantoic membrane of the chick embryos, researchers must cut a hole in the shell (in ovo), or gently crack the egg into a dish (ex ovo). Tumor vascularization and growth can be studied in three dimensions over the course of several days. Mitrevska et al used both in ovo and ex ovo CAM assays in to study.

Comparing the effectiveness of platinum nanoparticles against cisplatin

This new work evaluates platinum nanoparticles (PtNPs) in a breast cancer xenograft model and compares their effectiveness to cisplatin. In addition to studying these effects on tumor growth, the authors looked at the effects of cisplatin and PtNPs on tumor metabolism. Previous work suggests that PtNPs exert effects through a different mechanism than cisplatin, forming complexes with DNA polymerase rather than DNA bases.

The activity of PtNP-10 and PtNP-40 (two types of PtNPs) against primary tumors from a human breast cancer cell line were compared to the activity of cisplatin. The Azure c600 was used in a study of ex ovo cultures inoculated with fluorescently labeled breast cancer cells. The developing tumors were treated with each of the anticancer agents, and the embryos were fluorescently imaged using the c600. The Azure c600 used was equipped with three RGB fluorescence channels to best detect fluorescent biomolecules in the visible range, Cy2/Cy3/Cy5.

Green fluorescent macrovisualization of MDA-MB-231 migration and colonization in the chick embryos by ex ovo CAM assay captured using fluorescent imaging on Azure c600 imager
Figure 2a from Mitrevska et al. Fluorescent visualization of MDA-MB-231 migration and colonization in the chick embryos by ex ovo CAM assay using Azure c600. Used to evaluate the antitumor and antimetastatic activity of platinum-based drugs in association with the impact onamino acid metabolism. Imaged on the Azure c600. Licensed under CC BY 4.0.

The fluorescent images captured (Figure 2A) show metastasis of the cancer cells throughout the chick embryos. Metastasis is reduced in the embryos treated with PtNPs compared to the untreated embryos and those treated with cisplatin.

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 model organism imaging and 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.

Additional experiments further characterized the effects of PtNPs and cisplatin in the CAM assays. These experiments described metabolic effects and changes in expression of TCA cycle enzymes. The Azure c600 was then used to visualize EtBr gels to confirm RNA integrity (Figure S1) and check for amplicon size and primer dimers after PCR as part of the gene expression studies.

Chemiluminescent Western blot showing RNA integrity verified using a bleach gel visualized using Azure c600
Figure S1 from Mitrevska et al. Gel visualized using the Azure c600 to show the isolation of RNA. Licensed under CC BY 4.0.
Validation of the amplicon size and control of primer-dimer formation, visualized by Azure c600 from Azure Biosystems
Figure S2 from Mitrevska et al. Amplicon size and control of primer-dimer formation were checked by gel electrophoresis, and visualized using Azure c600. Licensed under CC BY 4.0.

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Conclusion

The authors concluded that the CAM assay is a powerful model to characterize anticancer drugs. The amino acid metabolism in MDA-MB-231 cell culture is the most susceptible to PtNPs-10. Their experiments demonstrate PtNPs are more effective than cisplatin at inhibiting tumor growth and metastasis and suggest a different mechanism of action for PtNPs than cisplatin.

SOURCES

  1. Mitrevska K, Rodrigo MAM, Cernei N, et al. Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism. Mater Today Bio. 2023;19:100570

Cielo Furthers Forensic Science Research at Saint Louis University

Categories
Customer Spotlight qPCR

Customer Spotlight: Erik Hall, Director of the Forensic Science Program at Saint Louis University (SLU)

At Saint Louis University (SLU), director of the Forensic Science Program, Erik Hall, provides students the most up-to-date methods for high quality analysis of DNA samples with data collected using the Azure Cielo 6 qPCR System.

Popularized by true crime shows and procedural dramas such as Bones, NCIS, and the CSI franchise, forensics has captured the public’s imagination. Forensic science is the application of scientific methods to matters of law; it draws on familiar fields, from biology to chemistry to computer science. Viewers tune in to watch investigators process evidence and triumphantly solve crimes. However, “unlike on CSI or these other shows, forensic scientists perform very specialized work (ie. someone is performing DNA and a different person the fingerprints, etc.),” explains Hall. “Forensic Science is an ever-changing field where you need to be on the cutting edge of technology and techniques.”

Erik Hall, Director of Forensic Science Program at SLU, using touchscreen on Azure Cielo qPCR System
Erik Hall (pictured) calls out the touch screen and the Cielo's intuitive interface as factors that help facilitate easier teaching moments and student collaboration efforts. In addition, the speed of the run, ease of initial setup, and the "great support staff" have been some of the most useful features of the Cielo.

The Cielo's role in the SLU forensic science program

After over a decade of working as a DNA Analyst and Biology Technical Leader for the St. Louis Metropolitan Police Department Crime Lab, Hall transitioned to teaching at SLU. At the Police Department, Hall shared a qPCR system with a local crime lab, but after his transition to SLU, found himself in need of his own.

What is qPCR?

Forensic students at SLU learn to process crime scene evidence for DNA, including testing to identify fluids, swabbing to collect fluids and cells, processing to extract DNA, and quantifying DNA. The University’s program prepares students for work in crime labs or crime scene processing environments, industries such as life sciences or pharmaceuticals, or graduate school. The Cielo 6 qCPR System was a great fit for students to see if there is enough material to obtain a genetic profile.

"The students have found the Cielo easy to use and adaptable to whatever kit we are using. The machine has performed as expected and is a great addition to our forensic science teaching and research lab at SLU,"

Identifying qPCR best practices for forensic processing using the Cielo

In his own research, Hall is focused on finding best practices and techniques when combining multiple analyses on a single item of evidence. Questions his lab investigates include when fingerprint and DNA analyses are both needed on the same itemwhat the best order in which the processes should be completed is, and can choosing specific techniques for each processing step improve the chances for success for all steps? “Fingerprint examiners and DNA analysts don’t always have these conversations. The hope of this research is to start more conversations between different sections within crime laboratories,” Hall says.

"Cost is always a concern with instrumentation. The Cielo was affordable while producing similar quality output to what I was used to seeing in the crime lab."

Today, Hall and his students use the Cielo to quantify extracted DNA, as DNA yield is the primary endpoint in some of their projects. “The qPCR machines are a crucial step in forensic science and one that gives us an accurate value of how much, if any, DNA we have to proceed in our processing,” he explains. For their studies, they choose to use forensic science–specific quant kits, such as the Powerquant kit from Promega, to run on the Cielo.

Check out this application note using the Cielo 6 and Promega GoTaq® Enviro Wastewater SARSCoV-2 System to detect SARS-CoV-2 in municipal wastewater

SLU Director of Forensic Science, Erik Hall, and undergraduate student, Anna Richards, with Azure Cielo qPCR System
Erik Hall (left) and SLU Forensic Science undergraduate student, Anna Richards (right), regularly use the Azure Cielo 6 to determine if using certain fingerprint chemicals can lead to more effective downstream DNA analysis, whether there are better DNA extraction methods to use after fingerprint chemicals have been added to a sample, and whether these DNA extraction methods can be expanded to other challenging samples submitted for DNA processing.

With the research being done with the Cielo at SLU, he hopes to spark more dialogue within crime laboratories. Unlike what is commonly seen on T.V. crime shows, real-life forensic scientists perform very specialized work. Hall’s hope is empirical data will prove the best practices to lead us to better, more robust answers. In turn, clearer answers give defense attorneys, prosecutors, and detectives better data to rely on when determining the outcomes of a criminal case. The forensic work being done at SLU aims “to make sure that the crime laboratories and others have the most up to date and best technology to ensure they provide the highest quality analysis for all cases.”

The Azure Cielo makes quantitative qPCR easy as ABC

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

Fingerprint methods used in crime labs and the field can make DNA extraction more challenging. The research being done with the Cielo 6 at SLU is creating new collaboration opportunities between disciplines across the forensic science field. His research has implications beyond just fingerprints and DNA- it raises the question of how various evidence processing methods affect the quantity and quality of DNA obtained from samples.

Forensic science is an ever-changing field that requires each moving part to be need to be on the cutting edge of technology. Hall’s mission with his research at SLU is to make sure crime laboratories and others have the most up to date and best technology to ensure they provide the highest quality data analysis for all cases. Check out what Hall and the forensic science students at SLU are up to by visiting their website here.

To learn more about the Cielo qPCR System and how it can fuel your thirst for data accuracy, try the Cielo free for 7 days.

 

More blog posts to read about qPCR: