Tag: Western blot

The Key to Early Alzheimer’s Detection

Categories
Western Blotting

Alzheimer’s disease is the most common cause of dementia and affects over 6 million Americans.1  It is important to research early indications of Alzheimer’s. This age-related, progressive disease is marked by the accumulation of protein aggregates in the brain (beta amyloid plaques around neurons and tau tangles within neurons) and by a decrease in brain glucose metabolism. Surprisingly, studies with subjects who were genetically predisposed to Alzheimer’s disease have found that those two neurological symptoms can begin to occur decades before Alzheimer’s symptoms become apparent. Currently, it is not possible to identify Alzheimer’s disease during this long preclinical period when interventions might be most effective.

Identifying biomarkers that may serve as early indicators of Alzheimer’s disease is an important subject of current Alzheimer’s research. The ideal identifying biomarker would be one that could be measured using inexpensive and noninvasive methods. To this end, Yao et al investigated in a recent paper whether mitochondrial dysfunction could be detected in extracellular vesicles (EVs) from the blood of Alzheimer’s patients. Given that EVs are small vesicles released into biological fluids like blood from many types of cells, they have been shown to contain analyzable components from the cells that release them such as nucleic acids and proteins.

Figure 1 from Yao et al. (2021) Mitochondrial electron transport chain protein abnormalities detected in plasma extracellular vesicles in Alzheimer’s Disease. The Azure Biomolecular Imager was used to image chemiluminescent blots analyzing markers present in the extracellular vesicle prep (panel B). Licensed under CC BY 4.0.

The authors collected EVs from the plasma of patients with high-probability early Alzheimer’s disease and from control patients with no evidence of dementia. A neuronal marker was used to selectively enrich for EVs from neuronal cells. The resulting EVs were characterized by Western blot to demonstrate that they contained EV markers and not markers associated with potential contaminants. To maximize image clarity and ensure accuracy, the chemiluminescent Western blots were imaged using the Azure Sapphire Biomolecular Imager.

Biochemical assays revealed that neuronally derived EVs from patients with Alzheimer’s disease had significantly lower levels of mitochondrial electron transport chain (ETC) complexes and lower levels of superoxide dismutase than EVs from control patients. In addition, the EVs from Alzheimer’s patients had significantly reduced activity of ETC complexes IV and V. The authors concluded that the neuron-derived EVs demonstrate the same abnormalities observed in Alzheimer’s tissues and model systems of Alzheimer’s disease and therefore may provide a biomarker for detecting early mitochondrial dysfunction in Alzheimer’s disease.

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

 

SOURCES

  1. Yao PJ, Eren E, Goetzl EJ, Kapogiannis D. Mitochondrial electron transport chain protein abnormalities detected in plasma extracellular vesicles in Alzheimer’s Disease. Biomedicines. 2021;9(11):1587.
  2. Alzheimer’s Association. 2021 Alzheimer’s Disease Facts and Figures. Alzheimers Dement 2021;17(3).
  3. Mustapic M, Eitan E, Werner JK, et al. Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017;11:278.

History Behind In-cell Westerns

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In-cell Western Western Blotting

Western blotting vs. In-cell Western blotting

Western blotting was first described by three groups in 1979. Since then numerous refinements to the basic technique, reagents used and imaging technologies have massively broadened the usage of Western blotting, making it a cornerstone protocol in life sciences today.

However, the general workflow of a Western blot has remained the same throughout this time. Samples are isolated and protein is extracted and standardized. Proteins are then separated by gel electrophoresis, transferred and probed with primary and secondary antibodies before being visualized. With numerous steps, Western blotting as a protocol requires a large investment in equipment, training, reagents and, perhaps most importantly, time.

A standard Western blot protocol takes two days to work from sample to image. Therefore, when analyzing a large number of samples or performing optimizations, it is easy to see how Western Blotting can quickly become a bottleneck in your research workflow.

in-cell Western with Azure Sapphire laser scanner
A serial dilution of HeLa cells were seeded into a 96-well plate, cultured, fixed and permeabilized. The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

What is In-cell Western blotting?

Enter in-cell Western blotting – a radical rethinking of the standard protocol coupling the ability to accurately quantify intracellular proteins from Western blotting with the repeatability, quick turnaround and high throughput of an ELISA. Cells are grown on sterile plates, before being fixed and permeabilized in situ. Subsequent labelling is performed as normal with a specific primary antibody followed by an isotype specific fluorescent conjugated secondary antibody. Conjugates in the near-infrared (NIR) spectrum are commonly used to reduce auto-fluorescence and noise typically associated with tissue culture plastics.

The Azure Biosystems Sapphire™ FL Biomolecular Scanner is specifically designed to allow for visualization in two NIR channels (658 nm and 785 nm) as well as visible fluorescent wavelengths (488 nm and 520 nm), allowing for multiplex imaging within wells. This multiplex capacity allows for the assessment of multiple proteins of interest in a single well which is useful for looking at total and phosphorylated versions of a protein or to detect an in-well standard to allow for quantification across a plate.

Azure Sapphire FL Biomolecular Imager with lid open
With a laser-based excitation system using photomultiplier tube (PMT) and avalanche photodiode (APD) detectors, signal selectivity, sensitivity and speed of imaging achievable by the Sapphire FL make it an ideal choice for in-cell Western blotting.
Full List of Sapphire FL Applications

In the application note below, we demonstrate the ability to quantify intracellular proteins in-situ using the Azure Sapphire. 

Materials and Methods

Culture cells

HeLa cells were serially diluted and seeded into a sterile 96- well tissue culture plate at a volume of 0.2 mL per well, and grown until approximately 80% confluent. All wells were fixed and permeabilized using 100% methanol for 15 minutes at room temperature.

In-cell Western blotting

Following fixation and permeabilization, cells were rinsed in PBS, blocked with 1% fish gelatin in PBS for one hour at room temperature, then probed for alpha-tubulin and beta-actin overnight at 4°C. Samples were washed three times with PBS prior to incubation with AzureSpectra 550 and AzureSpectra 800 conjugated secondary antibodies for 60 minutes.

Secondary antibodies were incubated along with RedDot™1 Nuclear Stain to normalize for total cell number. Plates were washed as previously described before imaging.

Imaging

After washing, the plate was imaged with the Sapphire using the Plate Focus Setting.

In- cell western performed using Azure Sapphire
Figure 2. A serial dilution of HeLa cells were seeded into a 96-well plate, cultured, fixed and permeabilized. A) Columns 1-3 were probed for beta-Actin using AzureSpectra 550 (green). B) Columns 3-6 were probed for Tubulin using AzureSpectra 800 (blue). C) The entire plate was stained with RedDot1 Nuclear Stain as a normalization control (red). D) The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

Results and Conclusions

HeLa cells were grown in a 96-well plate probed with alpha-tubulin and beta-actin antibodies followed by isotype appropriate secondary antibodies conjugated with AzureSpectra 550 and AzureSpectra 800 respectively. All wells were incubated with RedDot™1 Nuclear Stain to normalize for total cell number. Images were acquired using an Azure Biosystems Sapphire™ Biomolecular Scanner and are displayed in Figure 2. The representative image shown demonstrates the high level of sensitivity and specificity achievable.

Performing in-cell Western blotting on 96 well plates allows for accurate measurement of intracellular proteins expression in situ and provides a high throughput methodology to assess multiple stimulations, end-points, proteins of interest and replicates on a single plate. By using NIR antibodies and the Azure Sapphire the potential for in-well multiplex analysis also exists offering further improvements to throughput.

North Carolina’s Elite Christmas Tree Industry

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Customer Spotlight Imaging Western Blotting

Customer Spotlight: Adarsha Devihalli, PhD Candidate at North Carolina State

Nestled in the southern region of the Appalachian Mountains is an environmentally beneficial abundance of Fraser fir—the most sought-after Christmas tree in the USA. Thanks to its charming aroma, soft and durable needles, and eye-catching silhouette the tree forms the foundation of a multi-million dollar industry in North Carolina. It is these qualities combined with this unique geography that make North Carolina the second-leading Christmas tree producer in the United States. And while Fraser firs are heavily popular with holiday enthusiasts, they’re also extremely vulnerable to Phytophthora, a common cause of root rot disease.

"The Azure 400 Imager comes in [and is] a multi-user instrument…so we don’t have to run different instruments or look for labs that have all the instruments for us. Once I’m sure I’ve identified Phytophthora, I can use the cultures for my downstream experiments.”

Adarsha Devihalli
Adarsha and Dr. Whitehill standing next to azure c300
Adarsha and Dr. Whitehill with Azure 400 imaging system in their lab.

Several scientists at North Carolina State University are not letting this pathogen get in the way of Christmas tree production. For PhD student Adarsha Devihalli, the solution is in the molecular details. His research focuses on studying a particular strain of Phytophthora and its genetic code. His initial work focused on pathogen identification. In this next phase of his research, he will use functional genomics tools to enable the identification of genes in the pathogen important for the initiation of the infection process.

Devihalli isn’t the only one working on Phytophthora, either. He is a member of the Christmas Tree Genetics (CTG) Program, headed by Dr. Justin G. A. Whitehill, Assistant Professor and Director of the Christmas Tree Genetics Program at NC State University.

Under the guidance of Dr. Whitehill, Devihalli is studying this devastating disease to better understand the issues at hand. Together, Whitehill CTG lab members are working towards the development of novel genomic resources for Fraser fir to combat several pests of these celebrated trees.

Azure 400 Visible fluorescent imager
The Azure 400 is capable of three-channel visible fluorescence detection, which enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes. This fluorescent imager allows users to simultaneously image and quantify up to three different targets.
Learn More about the Azure 400

How the samples are collected

To begin his experimental process, Devihalli first visits the NC Department of Agriculture’s research station in Ashe County – located approximately four hours away from the university in Raleigh. He looks for disease-related symptoms on Fraser firs, collects samples, and returns to the lab for culturing, identification, and analysis using the Azure 400 Imaging System.

Looking to the future

Together, the Whitehill CTG lab and Devihalli intend to use their experimental results to help further current knowledge of the Fraser fir genome, and uncover potential genetic resistance mechanisms to Phytophthora root rot.  Ultimately, they plan to develop better mitigation methods for root rot in the country’s most beloved Christmas tree.

“At present, there is no publicly available sequencing information for these species,” explains Devihalli. “We don’t have a genome sequence for Fraser fir, so this is a big goal for our lab [yet].”

DISCOVER: Azure 400 Imager

For more information on Dr. Whitehill’s Christmas tree research at NC State, visit https://research.cnr.ncsu.edu/sites/whitehilllab/

Quantitative Western Blot Quiz

Categories
Western Blotting

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

• Follow your typical Western blotting protocol
• Be sure to probe for your protein-of-interest and a housekeeping protein so you can normalize your data
• Image the blot on a digital imager
• Draw boxes around the bands of your protein-of-interest and your housekeeping protein and use the imager to generate a number for band intensity
• Follow your imager’s instructions for subtracting background
• Calculate the ratio of your protein-of-interest to housekeeping protein to obtain relative protein abundance

What’s your quantitative western blotting IQ?

[qsm quiz=1]

More posts on Western blotting:

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

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

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

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

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

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

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

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What is Western Blotting?

Categories
Fluorescence imaging Quantification Troubleshooting Western Blotting

Your common Western blotting questions, answered.

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

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

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

Explore: Western Blot Imaging Systems

What is chemiluminescent detection?

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

What's more sensitive: chemiluminescence or fluorescence?

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

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

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

Continue reading: Beginning Chemiluminescent Western Blotting

Explore: Radiance Q

 

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

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

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

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

Is HRP a chemiluminescent substrate?

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

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

What is a chemiluminescence substrate?

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

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

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

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

Additional Western Blotting Resources

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Investigating the Mechanism of Pathology in Parkinson’s Disease

Categories
Imaging

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

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

The research compared 4 groups of mice:

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

DISCOVER: Azure 600

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

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

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

 

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How the Sapphire Scanner is Used to Better Grocery Store Tomatoes

Categories
Imaging Western Blotting

Few things are more disappointing than a tasteless, mealy grocery store tomato. These bland fruits are pale imitations of the vine-ripened tomatoes available from the garden at the end of summer. Tomatoes are perishable, and providing ripe, high-quality tomatoes that maintain their texture and nutritional content is a challenge for commercial growers.

A recent publication by Tsafouros et al from the Institute of Olive Tree in Greece provides a window into the intense, ongoing research aimed at understanding how and why tomatoes and other fruits ripen under various conditions. Such studies could improve the postharvest shelf-life and the quality of commercially grown tomatoes.

The role of temperature in mediating postharvest polyamine homeostasis in tomato fruit
The role of temperature in mediating postharvest polyamine homeostasis in tomato fruit

The authors carried out an exhaustive biochemical and molecular biological characterization of polyamine metabolism in the tomatoes. They assessed the total content of a variety of polyamines, the activity of the enzyme responsible for breaking down polyamines, the expression of all 23 genes encoding factors known to be involved in polyamine metabolism, the levels of the proteins involved in polyamine synthesis, and the levels of hydrogen peroxide, a biproduct of amine oxidases acting on polyamines. Protein levels were measured by chemiluminescent Western blots imaged using the Sapphire Biomolecular Imager from Azure Biosystems.

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The findings

Their results demonstrate that cold storage alters polyamine metabolism, and support storage of tomatoes at 10 °C after picking at commercial maturity. Lower temperatures appeared to induce a stress response, perhaps to protect against chilling injuries, while higher temperatures were associated with lower polyamine levels and lower quality fruit.

Growing the tomatoes used in this study

For this study, tomatoes were grown in a greenhouse and either picked at “commercial maturity” (when the tomato is just turning color) or left on the plant to mature for an additional week. The harvested tomatoes were stored at 5, 10, or 25 °C and after 7 days were compared to each other and to tomatoes left to ripen on the vine.

Tomatoes are typically picked before ripening and stored and transported at low temperatures in an attempt to increase the shelf-life. Tsafouros et al examined the effect on ripening of storing picked tomatoes at various temperatures for a week. The authors also characterized in detail the effect of storage temperature on the metabolism of polyamines, compounds known to play a role in fruit ripening and the content of which are known to be associated with tomato quality and shelf life.

Learn more about the Sapphire imager and how it can support your research by requesting a quote.

More published research using the Azure Sapphire:

Regulation of Gene Expression by Enhancer RNAs

Categories
Imaging Quantification Transfers Western Blotting

The regulation of gene expression is a complicated affair. A vast array of control mechanisms exist that can adjust the levels of gene expression products to match the needs of the cell. Messenger RNA (mRNA) can be processed to alter its stability, protein translation from mRNA can be controlled, and the stability and/or activity of the protein can be altered via post-translational modifications. The first point of control of gene expression is the initiation of gene transcription.

Setten et al studied an eRNA transcribed from an enhancer near the gene encoding a transcription factor called CEBPA (CCAAT enhancer-binding protein alpha). CEBPA is a transcription factor involved in many processes, including cell cycle inhibition and tumorigenesis, and expressed in specific cell lineage. It also plays a role in maintaining cell identity. To study CEBPA protein levels, the authors carried out a quantitative near-infrared fluorescent Western blot imaged on an Azure Sapphire™ Biomolecular Imager (Figure 7). To quantify changes in protein level between samples, the fluorescent signal from the Western blot was normalized to the total protein loaded, as was visualized on the Sapphire.

Figure 7 from Setten RL, Chomchan P, Epps EW, et al. (2021) CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA. Licensed under CC BY 4.0. Quantitative fluorescent Western blot showing levels of CEBPA isoforms detected with a goat–anti rabbit 800 secondary antibody from Azure Biosystems (red) and a NIR protein ladder (blue) (panel A). Before blocking the membrane was stained with a NIR fluorescent total protein stain and an image acquired for total protein normalization (panel B).​

The findings

The researchers set out to determine whether an eRNA transcribed from an enhancer 9kb downstream from the transcription start site of the human CEBPA gene was involved in regulating CEBPA expression. They called this eRNA CRED9. The authors found that levels of CEBPA mRNA and the CRED9 eRNA were correlated across several different cell lines; when CRED9 was high, CEBPA mRNA was also high. They then knocked down CRED9 in a cell line and found that when CRED9 levels were reduced, CEBPA mRNA and CEBPA protein levels were also reduced.

Finally, knockdown of CRED9 reduced the amount of a histone H3K27ac bound to the enhancer, indicating that the activity of the enhancer region was reduced. These results lead the authors to propose that CRED9 and other eRNAs may have an active role in enhancer function and gene regulation.

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Requirements of transcription initiation

Transcription initiation has several requirements. The chromatin structure must open to make the gene accessible to the transcriptional machinery. In eukaryotic cells, the promoter sequence of the gene must be bound by transcription factors that direct RNA polymerase to the gene to begin transcription. Transcription initiation is made more likely by the binding of activator proteins to other DNA regions near the promoter called enhancers, which can be located up- or downstream of the transcription start site.

Genome-wide sequencing experiments have revealed that RNA molecules are transcribed from many enhancer regions, indicating the enhancer regions may not merely be binding sites for activator proteins. These enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) and are not translated into proteins. It is possible that eRNAs may simply be the result of non-specific transcription by RNA polymerase and serve as a sign that chromatin is open and accessible to RNA polymerase in a region or DNA. Alternatively, there is evidence some eRNAs may serve an active role in regulating gene expression by themselves binding to and changing the activity of proteins.

In addition to multichannel and NIR fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor and white light imaging of blots, gels, tissues, and more. Download a free copy of the Sapphire Applications Booklet and learn about how you can add more applications to your arsenal here.

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Investigating S-acylation of SARS-Cov-2 Spike Protein Leads to New Insights into Viral Infectivity

Categories
COVID-19 Fluorescence imaging Imaging Western Blotting

A better understanding of coronavirus biology can enable development of new antivirals to help stop COVID-19 and prevent future pandemics. In recent work, Puthenveetil et al. characterized S-acylation of the Spike (S) protein of SARS-CoV-2. This post-translational modification is known to be important to the viral replication cycle of other viruses across multiple virus families but has not been studied in SARS-CoV-2.

To control for gel loading and S protein expression in the S-acylation experiments, the authors used the Azure Sapphire Biomolecular Imager to detect GAPDH by NIR fluorescence and SARS-CoV-2 S protein by chemiluminescence on Western blots (Figure 1).

Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay
Figure 1 from Puthenveetil R, Lun CM, Murphy RE, et al. (2021) S-acylation of SARS-CoV-2 Spike protein: mechanistic dissection, in vitro reconstitution and role in viral infectivity. Licensed under CC BY 4.0. The Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay (panel B).

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The authors expressed S protein in cultured cells and carried out metabolic labeling with a fatty acid that was detected using a rhodamine-labeled fluorescent probe. They found substantial S-acylation that was blocked by 2BP, a global inhibitor of S-acylation. The authors also found the S-acylation of the S protein was dependent on the presence of the cysteines in the C-terminal domain.

Further experiments expressing mutated versions of the S protein identified which cysteines were S-acylated, the effect of mutating these cysteines on particle infectivity, and which members of the human family of enzymes that carry out S-acylation were able to modify the S protein in cells and in an in vitro assay.

What is S-acylation?

S-acylation involves adding long-chain fatty acids to cysteine residues on the cytosolic side of transmembrane proteins. The cytoplasmic tail of the SARS-CoV-2 S protein contains 10 cysteines in 6 potential S-acylation sites. Puthenveetil et al note that all but one of these are conserved with SARS-CoV, and most are conserved with other coronaviruses that infect humans, including MERS. Still, nothing is known about what role, if any, the S-acylation of these cysteines may play in the biology of viruses, such as SARS-COV-2.

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

More research done with the Azure Sapphire:

Problems with Ponceau Stain? Consider Alternative Total Protein Stains for Fluorescent Western Blots

Categories
Fluorescence imaging Troubleshooting Western Blotting

Keep reading if you’re having problems using ponceau! Ponceau S is a fast, reversible protein stain often used to confirm that protein samples have successfully transferred from the gel to the membrane before a researcher moves ahead with the time-consuming process of immunoblotting.

Staining the membrane after transfer makes it possible to quickly and easily identify any problems such as incomplete or uneven transfers, or artifacts due to the presence of air bubbles, before probing the blot with antibodies. In addition, staining the blot with some total protein stains can provide a standard for total protein normalization of quantitative Western blots.1

Even though Ponceau staining is reversible, it is not compatible with fluorescent Western blot detection. After thorough destaining, Ponceau stain can leave an autofluorescent residue on the membrane that increases background fluorescence.

  • Azure Quick Tip: To avoid high background due to Ponceau staining, consider using other total protein stains.

    AzureRed Fluorescent Total Protein Stain is completely compatible with downstream Western blotting detection, including fluorescent detection, and with downstream mass spectrometry.

Learn more about AzureRed Fluorescent Total Protein Stain

The figure below shows a multicolor fluorescent Western blot. The right half of the blot was stained using Ponceau and then destained before immunoblotting.

High background when fluorescent western blot stained with ponceau

Both halves of the blot were then blocked with Azure Fluorescent Blot Blocking Buffer and probed for four proteins using four different fluorescent probes as labeled in the figure. The blot was imaged using the Azure Imager RGB module which assesses Cy2, Cy3, and Cy5-compatible channels. Despite thorough destaining, a very high fluorescent High background when NIR western blot stained with ponceaubackground is seen on the half of the blot that was stained with Ponceau.

The figure to the right shows the same blot imaged using the Azure cSeries NIR module, which assesses 700nm and 800nm channels. The fluorescent background is reduced in the NIR-imaged blot, but is still substantial compared to the unstained half of the blot.

Additional blog posts on total protein:

Shop Ponceau Stain Alternatives, like Azure Ponceau Stain

SOURCES

  1. Thacker JS, et al. Total protein or high-abundance protein: Which offers the best loading control for Western blotting? Biochem. 2016; 496:76-78. PMID: 26706797.
  2. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. 2017;17:1600189. PMID: 28941183.