Let’s Talk about Real-time PCR! Part 2

Categories
qPCR Troubleshooting

This is part two of our “Let’s Talk about Real-time PCR” post, so check out part one if you haven’t yet.

Are you seeing strange or unexpected results in your quantitative PCR (qPCR) reactions?  Here are some commonly experienced issues, and some possible solutions to try.

 

1. Why is my PCR efficiency so low?

The efficiency of a PCR reaction is the fraction of target molecules copied every PCR cycle. In general, an efficiency of at least 90% is recommended. Many factors can contribute to a lower efficiency, including inefficient annealing between the primers and target, the primers binding to competing sites, the presence of inhibitors in the sample, and insufficient reagents in the mix. Double check the primer sequences to make sure there are no potential competing reactions. Run a melting curve to make sure you aren’t amplifying unexpected products. Reaction conditions may need to be optimized.

 

2. My PCR efficiency is too high! How is it possible to have an efficiency greater than 1?

A PCR efficiency greater than 1 would suggest that more than 100% of the targets are replicated each cycle. What’s going on? The presence of inhibitors in the sample is frequently the source of efficiency measures greater than 1. The greatest inhibition is in the most concentrated samples used in a dilution series, so the effect of inhibition on the standard curve is more pronounced at one end and distorts the slope of the curve, changing the efficiency calculation. You may need to dilute the sample to dilute out any contaminants.

 

3. Why are my Ct values so low? There’s no way there was that much target in my sample.

Your samples may have evaporated if they were not stored correctly, increasing the concentration of the target. Carry out a melting curve at the end of PCR to make sure you are only amplifying the expected target and not amplifying something unexpected or primer dimers.

 

4. Why is there amplification in the no template control?

Contamination is a major consideration when carrying out PCR. Make sure your pipettes and workplace are clean so you are not potentially transferring amplified products from a previous experiment into your solutions. Also, run a melting curve to see if you are amplifying primer dimers.

 

We hope this is helpful as you troubleshoot your qPCR. Find more qPCR tips and solutions in the free Azure qPCR Troubleshooting guide.

Let’s talk about real time PCR! Part 1

Categories
qPCR

What is qPCR and how is it used in practice? Quantitative real-time PCR is a powerful technique for showing not only whether a nucleic acid sequence is or is not present in a sample, but for determining exactly how many copies of that sequence are present in the sample. PCR transformed biological research, and now, during the COVID pandemic, more people are talking about PCR than ever before.

So what is it? How is it used? And where do you turn when your reactions aren’t working how you expected them to?

This will be a two-part blog series. In Part I, let’s define some terms:

PCR: Polymerase chain reaction. The development of this technique to amplify specific pieces of DNA won its inventor the 1993 Nobel Prize in chemistry. PCR takes advantage of the double-strandedness of DNA. The DNA in a sample is sequentially heated to separate the strands, cooled to allow 2 primers, each specifically designed to bind to one strand bracketing the sequence to be amplified, to anneal to the separated DNA strands, and then incubated with a DNA polymerase which binds to the annealed primers and synthesizes the complementary DNA strands. At the end of each cycle, one piece of double-stranded DNA has been copied into two double-stranded pieces. Over subsequent PCR cycles, each copy is itself copied, so the number of copies grows exponentially.

Because PCR is amplifying the target sequence exponentially, if you start with a single piece of DNA, after 20 cycles of PCR, there will be over a million copies of the target sequence. PCR can be used to make enough copies to visualize on a gel, to construct recombinant plasmids, to use as probes in new experiments, to use in a diagnostic test, or other downstream applications.

RT-PCR: RT-PCR is “reverse transcription PCR”.  What if you want to detect or quantify RNA, which is single stranded?. The answer is RT-PCR; RNA is reverse transcribed (made into DNA) and then that DNA is subjected to PCR. Luckily, almost all messenger RNAs (mRNAs) that encode proteins have poly-A tails. So, by using a poly-T primer, all messenger RNAs can be reverse-transcribed into their complementary DNA sequence (cDNA).

Real-time PCR: In real time PCR, the accumulation of PCR products is followed in real time by incorporating fluorophores into the product and monitoring the fluorescence of the reaction.  There can be confusion around the term RT-PCR; convention is that RT-PCR stands for reverse transcription PCR and not real time PCR.

Ct value: The Ct value is the “cycle threshold” value. When watching fluorescence increase as PCR products accumulate in real time, initially the fluorescence signal will be too low for the detector to pick up. Scientists select a threshold value above which the fluorescence signal can be confidently detected. To build a standard curve, they see how many cycles it takes for a reaction with a known starting amount of target sequence to reach the fluorescence threshold value. That cycle number is Ct.

qPCR: qPCR is quantitative PCR. Real time PCR allows quantitation of the amount of target in a sample. Comparing the increase in fluorescence signal of the sample to a standard curve made by amplifying known starting amounts of the target sequence, the amount of target in an unknown can be calculated.

In future blogs, we’ll discuss PCR applications and troubleshooting.

Learn more about qPCR applications and Azure’s Real-time PCR instrument, the Cielo by clicking here.

Potential Treatment for Advanced Kidney Cancer

Categories
Protein Assays Western Blotting

Renal cell carcinoma (RCC), the most common type of kidney cancer, is aggressive and frequently develops resistance to therapy. Advanced RCC has a poor prognosis and new, effective treatment strategies are badly needed. Metastatic RCC may be treated with sunitinib, but the majority of cancers eventually develop resistance to this drug. Sunitinib inhibits signaling through receptor tyrosine kinases, interfering with pro-growth signals received by the tumor.

In a recent publication, Markowitsch et al investigated the effect of shikonin (SHI) on sunitinib-sensitive and sunitinib-resistant RCC cell lines in cell culture. SHI is a naturally occurring compound. It’s an active component of a dried plant root (Lithospermum erythrorhizon) that has been used in traditional Chinese medicine to address a variety of ailments.

Earlier studies have demonstrated the anti-cancer capabilities of SHI and have shown that SHI can enhance the activity of traditional chemotherapeutics or re-sensitize chemotherapy-resistant cells to therapy. How SHI exerts these effects is not clear; however, as SHI has been found to affect many cell signaling pathways and induce cell death via apoptosis and necroptosis.

The recent work by Markowitsch et al thoroughly examined the effect of SHI on many aspects of RCC cell biology. Several assays relied on imaging with the Azure Sapphire Biomolecular Imager, including studies that characterized protein expression related to multiple signaling pathways by Western blot, adhesion of RCC cells to extracellular matrix proteins and to vascular endothelial cells, and studies of tumor cell migration and chemotaxis, relied on imaging with the Sapphire.

Figure 2 from Markowitsch et al. (2022) Shikonin inhibits cell growth of sunitinib-resistant renal cell carcinoma by activating the necrosome complex and inhibiting the AKT/mTOR signaling pathway. Licensed under CC BY 4.0. The Azure Biomolecular Imager was used to image and quantify RCC colonies on cell culture dishes.

The authors took advantage of several imaging modes provided by the Sapphire. The Sapphire was used to image and quantify the growth of colonies in 6-well culture dishes using Coomassie Blue dye detection. The Sapphire was used to assess cell adhesion, chemotaxis and cell motility by measuring the fluorescence of cells labeled with CellTracker Green in either pre-treated 24-well culture dishes or on the lower surface of membrane inserts in 24-well plates. Western blots were detected using enhanced chemiluminescence and imaged on the Sapphire.

The results of the numerous studies indicated that, though the specific effects varied by cell line, SHI had antitumor effects on all cell lines studied. SHI was found to inhibit RCC cell growth, proliferation, and clone formation, both in sunitinib-sensitive and sunitinib-resistant cell lines. SHI caused cell cycle arrest and induced cell death, primarily via necroptosis. SHI also inhibited the AKT/mTOR pathway, which presents another mechanism by which SHI may interfere with RCC cell survival and growth.

The authors conclude the data is promising and that SHI should be further studied as a potential addition to therapy for patients with advanced and therapy-resistant RCC.

In addition to visible and fluorescent imaging of tissue culture plates, and chemiluminescent imaging of Western blots, 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 Imager and how Azure can support your research by clicking here.

 

Explore: Azure Sapphire Biomolecular Imager

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.2

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

 

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. Alzheimer’s Association. 2021 Alzheimer’s Disease Facts and Figures. Alzheimers Dement 2021;17(3).
  2. 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.
  3. 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.

Help! Why do my Western blots look terrible?

Categories
Western Blotting
One of the most common questions when troubleshooting problematic Western blots is “why is the background so high?”
 

High or uneven background on Western blots doesn’t just look bad- it interferes with data analysis, making it difficult to quantify bands or compare bands between samples.

There are several things you can do to reduce background and increase the signal-to-noise ratio on your blots. Read on for steps to help you achieve high-quality data and publication-worthy images!

5 Steps to reducing Western blot background
 
1. Use clean, fresh buffers.

Make sure your blotting and wash buffers are made fresh. You may want to filter them to remove dust or particulates that may be deposited on your blot and interact with your antibodies or other components of the blotting protocol.

 
2. Use the correct blocking agent.

Make sure you select a blocking agent that doesn’t interact with your antibody or block your epitope! Commonly used protein-based blocking agents can be problematic in specific situations, particularly with anti-phosphoprotein antibodies.
Read more about finding the best blocking agent for your application here.

 
3. Don’t skimp on the wash steps.

Make sure you use sufficient wash buffer, wash for a long enough time, and agitate the membrane well during wash steps. Any non-specifically bound antibody left on your blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer.

 
4. Find the best exposure time.

When over-exposed, any blot can appear as solid background. Ideally, the signal from specific bands is much stronger than any background noise and a short exposure will pick up only the specific signal. If using film, be prepared to expose the blot multiple times to different pieces of film for increasing periods of time to find the optimal exposure. Imaging with a CCD camera makes capturing multiple exposure times even easier. If using an ECL detection system, find a detection reagent with a stable, long-lasting signal such as Radiance ECL so exposure times are predictable and reproducible, and the signal doesn’t decay so rapidly that you cannot conduct multiple exposures.

 
5. Optimize your antibody concentrations.

This is a situation where some initial work up front can save you a lot of time down the line. Using too much antibody can increase the amount of antibody that binds non-specifically to the membrane. Start with the antibody dilution recommended by the antibody provider. If background is high, dilute the antibody more, increasing the incubation time if necessary. Incubating at 4 °C can also help reduce non-specific binding.

Reducing background on fluorescent western blots:

In addition to the steps mentioned above, for fluorescent blots there are a couple of additional things to try:

1. You may need to change your membrane.

Nitrocellulose and some PVDF membranes can autofluoresce. To reduce background from your membrane, use only low-fluorescence PVDF membranes.

2. Wet membranes also can autofluoresce.

Dry the membrane completely before imaging fluorescent blots.

3. Control the temperature during the transfer step.

Excessive heat during transfer is usually a major source of background in fluorescent Western blotting.



With these tips, you can reduce the background and get clean, clear Westerns.

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

    Categories
    Fluorescence imaging Quantification Troubleshooting Western Blotting
    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

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

    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.

    To avoid high background due to Ponceau staining, consider using other total protein stains.2 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

    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.

    Sapphire Used in Study of Green Tea’s Anti-cancer Effects

    Categories
    Fluorescence imaging Imaging

    Recent work published by Zhao et al in nature communications lends insight into a potential mechanism for the anti-cancer activity of green tea.

    The health benefits of drinking tea, particularly green tea, has been the subject of intense investigation. A large number of epidemiologic studies have examined whether tea consumption can reduce cancer risk, with a focus on a category of polyphenols called catechins, the most active and abundant of which in green tea is epigallocatechin-3-gallate (EGCG). According to the National Cancer Institute, EGCG and other polyphenols found in green tea have multiple biological activities including being potent antioxidants, inhibiting tumor cell proliferation, inducing tumor cell apoptosis, inhibiting angiogenesis, and activating detoxifying enzymes.

    In in vitro studies, EGCG has been shown to induce apoptosis in cancer cell lines. The transcription factor p53 plays a role in this activity of EGCG. P53 is an important tumor suppressor, promoting cell cycle arrest or apoptosis when a cell is stressed. Usually, cellular p53 levels are low, with p53 being ubiquitylated which targets the protein for degradation. The ubiquitylation is carried out by the protein MDM2, an E3 ligase. When cells are stressed, ubiquitylation is suppressed, p53 is not degraded, and p53 levels increase. The p53 protein then promotes apoptosis through interactions with other proteins and by inducing gene expression changes. Earlier studies have shown that EGCG stabilizes p53 and may interfere with the interaction of p53 and MDM2.

    In their current study, Zhao et al demonstrate that EGCG binds to the N-terminal domain of p53. They also show that EGCG disrupts the interaction of p53 with MDM2 and inhibits ubiquitylation of p53 by MDM2. In the publication, ubiquitylation assays were carried out using fluorescein-labeled p53 and varying amounts of EGCG. After incubation with MDM2 and ubiquitin, the amount of free and ubiquitinated p53 was detected on SDS-PAGE gels using the fluorescein channel of a Sapphire Biomolecular Imager. The amount of ubiquitin was quantified and concentration-dependent inhibition of ubiquitylation by EGCG was analyzed.

    The authors propose that the EGCG-induced reduction in ubiquitylation could stabilize p53 protein, making more p53 available and contributing to its anti-tumor activity. Additionally, the work suggests the N-terminal domain of p53 presents an attractive target for anti-cancer drug design.

    The Sapphire imager provides multiple imaging capabilities in addition to multi-channel fluorescence, including white light, phosphor imaging, and chemiluminescence imaging of blots, tissues, microplates, and more. Learn more about the Sapphire imager and how it can support your research by clicking here.

    Western Blotting Reagents Roundup – Recent Publications

    Categories
    Controls Multiplex Western Blotting

    Azure offers products to support every step of your Western blotting workflow, from blocking to detection. Several recent publications highlight the use of Azure reagents in chemiluminescent and fluorescent Western blotting, in a variety of experimental systems and approaches. Below is a brief summary of four articles in which researchers incorporated Azure’s protein-free blocking buffer, secondary antibody conjugates, and ECL detection reagents into their research.

    Adipocyte REVERBα dictates adipose tissue expansion during obesity

    In a recent pre-print, Hunter et al recently demonstrated that REVERBa, a circadian clock component, modulates white adipose tissue metabolism in response to changes in metabolic state. The researchers created a mouse model in which REVERBa was knocked out in only white adipose tissue and compared these mice to mice missing REVERBa in all tissues. Chemiluminescent and fluorescent Western blots were used to confirm protein changes in knock out mice, and to follow circadian changes in protein levels. All Western blot experiments used Azure’s Protein-Free Blocking Buffer to block nitrocellulose membranes. This blocking buffer is a great choice to enhance signal and reduce background for general Western blots and is compatible with all detection chemistries.

    Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma Using a Cell-Penetrating Peptide

    Smith et al identified a novel potential therapeutic approach for pancreatic cancer. In Molecular Therapy Oncolytics, the researchers examined whether a peptide that mimics a part of the proliferating cell nuclear antigen (PCNA) could interfere with PCNA-protein interactions in pancreatic cancer cells. They found that the peptide killed cancer cells by inhibiting DNA replication and DNA repair, resulting in the accumulation of DNA damage. Fluorescent Western blots were used to assess DNA damage by detecting phosphorylated histone H2AX, a biomarker for double stranded DNA breaks. The secondary antibody used was the Azure Spectra 800 Secondary Fluorescent Antibody.

    Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs

    Cantu et al identified a surprising new function of vaccinia virus decapping enzymes. The decapping enzymes D9 and D10 are known to promote degradation of viral and cellular mRNAs. In PLOS Pathogens, the researchers compared genome-wide translation efficiency in cells infected with wild type vaccinia virus vs mutant virus lacking functional decapping enzymes. The results indicated that paradoxically, while promoting degradation of mRNAs, the decapping enzymes are also required for selective translation of certain post-replicative viral proteins whose mRNAs have 5’-poly(A) leaders. Translated proteins were detected on chemiluminescent western blots using Azure HRP-conjugated Secondary Antibodies.

    2‐Oxothiazolidine‐4‐carboxylic acid inhibits vascular calcification via induction of glutathione synthesis

    Patel et al are investigating the potential of a cysteine prodrug, 2‐oxothiazolidine‐4‐carboxylic acid (OTC) to inhibit arterial medial calcification. In a recent article published in the Journal of Cell Physiology, the researchers used Azure’s Radiance ECL HRP substrate to detect markers of cell differentiation in cultured vascular smooth muscle cells (VSMC), grown in a calcifying medium with or without OTC. Western blotting revealed that treatment with OTC reversed the increase in osteoblast markers and rescued the drop in glutathione synthesis enzymes observed in calcifying cells, leading the authors to conclude the drug was effective in vitro and might have clinical relevance. The long-lasting signal and high sensitivity of Radiance ECL substrate allows for reliable comparisons of protein levels between samples.

    You can find more publications using Azure reagents and imaging systems on the Azure publications list, or contact Azure for assistance identifying publications using a specific product.

    Studying the SARS-CoV-2 proteases with the Sapphire Biomolecular Imager

    Categories
    Fluorescence imaging

    Researchers at the Wroclaw University of Science and Technology and their colleagues have recently published exciting work improving our understanding of the two proteases encoded by the SARS-CoV-2 genome, the main protease (Mpro) and the papain-like protease (PLpro). These proteases are required for viral gene expression and for viral replication, so they are attractive targets for antiviral drug development. In addition, PLpro can inhibit the host innate immune response via deubiquitination activity, so inhibition of this protease may provide another mechanism of antiviral activity.

    In each publication, the scientists used hybrid combinatorial substrate libraries to understand the rules governing substrate specificity of these proteases. Then, these rules guided the design of activity-based probes and irreversible inhibitors for each protease.

    To characterize binding of their compounds to the proteases and the deubiquitinase activity of PLpro, the authors took full advantage of the fluorescence detection capabilities of the Sapphire Biomolecular Imager.

    In Science Advances, the group examined SARS-CoV-2 PLpro, designing and characterizing two inhibitors. Using a biotinylated ubiquitinated probe, they found no evidence for cross-reactivity between their inhibitors and human deubiquitinases, an important requirement for potential antiviral drugs targeted to this protease. The biotinylated probe, bound to proteins in cell lysates, was detected on blots using an Alexa Fluor 647¬–labeled streptavidin as a probe and imaging the blots with the Sapphire Imager. To study the deubiquitinase activity of SARS-CoV-2 PLpro, binding and cleavage of ubiquitin-containing substrates was detected in SYPRO Ruby-stained gels, also imaged using the Sapphire Imager.

    In Nature Chemical Biology, the group focused on SARS-CoV-2 Mpro, designing an inhibitor that demonstrated significant antiviral activity against SARS-CoV-2 in a cell culture assay, and an activity-based fluorescent probe that could be used to visualize active Mpro in cells taken from patients with COVID-19 infection. To study the binding of their compounds to SARS-CoV-2 Mpro, compounds labeled with Cy5 or BODIPY were detected directly on blots. Biotinylated versions were detected using Alexa Fluor 647¬–labeled streptavidin as a probe.

    This research spotlights numerous avenues for development of potential anti-coronavirus drugs and tools for disease characterization and diagnostics.

    With up to four solid state lasers as excitation sources, the Sapphire Biomolecular Imager provides the flexibility to detect a wide variety of fluorescent dyes, including near infrared. To learn more about the Sapphire and how it can improve your fluorescence imaging, contact us at info@azurebiosystems.com.

    https://doi.org/10.1126/sciadv.abd4596
    https://doi.org/10.1038/s41589-020-00689-z

    Gruber, Christian C.; Steinkellner, Georg (2020): Comparative model of novel coronavirus 2019-nCoV protease Mpro. figshare. Dataset. https://doi.org/10.6084/m9.figshare.11752752.v5 licensed under CC BY 4.0.

    Studying tissue morphology with the Sapphire Biomolecular Imager

    Categories
    Fluorescence imaging
    Sheep kidney imaged using 488nm and 658 nm lasers, 10 micron resolution

    The Sapphire Biomolecular Imager can do so much more than image gels, blots, and microwell plates. With its 25 cm x 25 cm scanning bed, the versatile Sapphire can scan tissues and even small animal models such as mice, zebrafish, and Xenopus oocytes, to study tissue morphology or gross anatomy.

    Bakela et al took advantage of this capability of the Sapphire Imager to study liver morphology in a recent publication. The group investigated the ability of soluble major histocompatibility complex II (sMHCII) molecules to rescue symptoms of autoimmune hepatitis (AIH) in a rat model of the disease.

    Chronic AIH is characterized by a T-cell-mediated autoimmune response that attacks the liver. The disease is usually treated with immunosuppressive drugs. New and specific therapies are needed to better treat the disease and to avoid the side effects associated with long-term use of immunosuppressants.

    The authors set out to test whether sMCHII molecules could rescue liver damage in a rat model of AIH. These molecules are hypothesized to help maintain immune tolerance and promote immune system suppression, protecting against autoimmunity. Promisingly, sMCHII molecules had been tested previously in a model of systemic lupus erythematosus and found to decrease the amount of autoantibodies and improve symptoms.

    To characterize the liver damage that occurred in the AIH rat model, the authors collected and fixed livers from the rats and then scanned them on a Sapphire Biomolecular Imager using white light as well as four-channel fluorescence. The four-channel images, detecting tissue autofluorescence, provided detail of the gross anatomy and morphology of the liver tissue. Treatment with sMCHII appeared to rescue the fibrotic and necrotic changes that were observed in the livers of untreated rats, leading the authors to propose this approach could lead to new therapies for AIH.

    Learn more about applications of the Sapphire Biomolecular Imager, including scanning tissues and small animal models using fluorescence, chemiluminescence, and phosphorimaging, here.