Western Blotting Reagents Roundup – November 2022

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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. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

The role of the epithelial to mesenchymal transition in cancer drug resistance and recurrence

In recent work, Ingruber et al1 hypothesized that head and neck squamous cell carcinoma (HNSCC) cells are in a partial EMT state, able to switch towards epithelial or mesenchymal phenotypes depending on environmental stimuli, and that this switch contributes to their proliferation and resistance to Cisplatin therapy.

As part of this work, the authors carried out chemiluminescent and near-infrared (NIR) fluorescent Western blots to assess levels of EMT protein markers. The authors used Radiance Plus substrate for chemiluminescent Western blots, and fluorescent secondary antibodies for the near-infrared blots. Western blots were then imaged using an Azure c500 imager. The work found that a partial EMT-like pathway appears to contribute to Cisplatin resistance in the cell line used, and that overexpression of an epithelial marker sensitized cells to Cisplatin while reducing a pro-EMT transcription factor. The results suggest future avenues to research and treat drug-resistant cancers.

Multicolor near-infrared Western blots and a combined chemiluminescence and NIR blot imaged using Azure 500 Western blot imager
Figure S2 from Ingruber J et al (2022). Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Licensed under CC BY 4.0. Multicolor NIR Western blots (panels A,B,D,E) and a combined chemiluminescence and NIR blot (C) were imaged on an Azure C500 imager.

The epithelial to mesenchymal transition (EMT) is a reversible process in which epithelial cells undergo biochemical changes to adopt a mesenchymal cell phenotype with increased ability to migrate and increased resistance to apoptosis. The EMT can play a role in normal processes such as embryogenesis and wound healing but also contributes to cancer metastasis and tumor cell migration.

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Lipid peroxidation in sporadic Alzheimer's disease

In a recent publication, Ramsden et al2 propose a new hypothesis for the mechanism behind sporadic Alzheimer’s disease (AD) in which the initiating factor of AD is lipid peroxidation of the apolipoprotein E protein (ApoE) and of the ApoE receptor. AzureRed total protein stain was used to detect total protein before immunoblotting. The blots were blocked with Fluorescent Blot Blocking Buffer and imaged with the Azure Sapphire Biomolecular ImagerThe peroxidation is hypothesized to disrupt important processes required for memory formation and maintenance of structural integrity, initiating a cascade that leads to AD. The proposed mechanism differs from the amyloid cascade hypothesis and would have important implications for AD prevention and therapeutics if confirmed. Lipid peroxidation is proposed to occur at the ligand-receptor interface of ApoE and the ApoE receptor where there are amino acid residues predicted to be susceptible to peroxidation.

Because polyunsaturated lipids are transported by ApoE, the ApoE-ApoE receptor interface may create a microenvironment favorable to lipid peroxidation. The hypothesis accounts for several observations about AD including the anatomic areas of the brain known to be affected, the fact that ApoE variants are associated with sporadic AD, that ApoE is enriched in neurite plaque cores, the significance of amyloid plaques and neurofibrillary tangles, and evidence that lipid peroxidation occurs very early in sporadic AD. To test their hypothesis, the authors conducted fluorescence immunoblotting to detect lipid aldehyde-induced crosslinking of ApoE and the ApoE receptor ApoER2.

Based on these in vitro experiments and additional experiments including immunohistochemistry of human brain samples, the authors conclude that their hypothesis is consistent with experimental observations and deserves additional study.

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A study of the anti-inflammatory effects of LRP1 ligands

Mantuano et al4 used 3 ECL substrates (Radiance, Radiance Q, and Radiance Plus) from Azure Biosystems in their investigation of the anti-inflammatory action of three ligands of LDL receptor protein-1 (LRP1). Chemiluminescent Western blots imaged on the Azure c300 or on film were key to the study as the authors assessed the components required for enzymatically-inactive tissue-type plasminogen activator (El-tPA), activated α2-macroglobulin (a2M), and a soluble derivative of nonpathogenic cellular prion protein (S-PrP) to activate signal transduction in macrophages.

The results found indicate that lipid rafts and the N-methyl-D-aspartic acid (NMDA) receptor are required by all three ligands studied, while LRP1 was not required by two of the ligands when the ligands were present at high concentrations. In addition to the effects on cell signaling, the ligands studied were also shown to prevent lipopolysaccharide (LPS)-induced shedding of LRP1. Since the soluble LRP1 product is pro-inflammatory, blocking this process is another way LRP1 ligands could convey an anti-inflammatory effect. The differences uncovered between the three ligands’ requirements for signal transduction activation might help clarify their effects on macrophages in various states of differentiation 

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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 on the left.

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SOURCES

  1. Ingruber J, et al. Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Biomedicines. 2022;10(10):2482.
  2. Ramsden CE, et al. Lipid peroxidation induced ApoE receptor-ligand disruption as a unifying hypothesis underlying sporadic Alzheimer’s disease in humans. J Alzheimers Dis. 2022;87(3):1251-1290.
  3. Mantuano E et al. The LRP1/CD91 ligands, tissue-type plasminogen activator, a2-macroglobulin, and soluble cellular prion protein have distinct co-receptor requirements for activation of cell-signaling. Sci Rep. 2022;12(1):17594.
  4. Jäntti MA, et al. Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2022;1867(11):159219.

New tools created for the study of coagulation pathway proteases

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Fluorescence imaging Imaging

In recent work, Modrzycka et al from Wrocław University of Science and Technology reported the development of novel activity-based probes (ABPs), inhibitors, and substrates specific to three serine proteases important to the blood coagulation pathway. The new reagents make it possible to differentiate these highly related proteases and study them simultaneously in complex biological samples. 

Fluorescent ABPs and covalent inhibitors

To allow real-time detection of protease activity, the authors created ABPs labeled with fluorescent dyes (Cy3, Cy5, or Cy7) instead of biotin. The probes served as covalent inhibitors of the proteases. Versions of the covalent inhibitors without the fluorescent probes were also characterized. To demonstrate specificity in vitro, purified proteases were incubated with the fluorescently labeled ABPs, run on a gel, transferred to membrane, and fluorescence imaged using an Azure Sapphire Biomolecular Imager.

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Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma. Chem Sci. 2022;13(23):6813-6829. Licensed under CC BY-NC 3.0. This panel shows three fluorescently labeled activity-based probes, designed to be specific for the three coagulation factors APC, fXa, and thrombin (fiiia), detected in-gel bound to their respective targets in human plasma. Direct in-gel imaging was performed at 520 nm for Cy3, 658 nm for Cy5, and 784 nm for Cy7 with the Azure Biosystems Sapphire Biomolecular Imager
Figure C from Modrzycka et al. Figure FC. Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma. Chem Sci. 2022;13(23):6813-6829. Licensed under CC BY-NC 3.0. This panel shows three fluorescently labeled activity-based probes, designed to be specific for the three coagulation factors APC, fXa, and thrombin (fiiia), detected in-gel bound to their respective targets in human plasma. Direct in-gel imaging was performed at 520 nm for Cy3, 658 nm for Cy5, and 784 nm for Cy7 with the Azure Biosystems Sapphire Biomolecular Imager.

Simultaneous detection of coagulation factors in human plasma

The authors then demonstrated that all three proteases could be detected individually in a complex biological sample using their specific fluorescent ABPs. Human plasma was incubated with each labeled probe and then separated on a gel. The gel was directly scanned on the Sapphire to detect each of the three fluorescent labels. As seen in figure 6C above, each of the three fluorescent probes is specific for its target protease. All three proteases could be detected simultaneously in the sample.

The authors conclude the newly developed tools may be used in multiple ways including the study of these proteases, pharmacologic knockdown of individual proteases, and diagnostics.

Characterization of protease substrate specificity

Modrzycka et al used libraries of peptides including natural and non-natural amino acids to probe the substrate specificities of APC, thrombin, and factor Xa at each of four amino acid positions in the protease cleavage site. Peptide substrates were then designed based the results, incorporating amino acids at each of four sites to try to obtain substrates that were specific to each protease. The authors successfully identified substrates that were efficiently hydrolyzed by each protease while demonstrating little or no detectable hydrolysis by the other two proteases.

Activity-based probes to study coagulation factors

Until now, it has been difficult or impossible to differentiate between active and inactive forms of these proteases in situ. Antibodies are usually unable to distinguish between the zymogen and the active protease. Therefore, the authors created biotin-labeled versions of their specific substrate sequences. These activity-based probes can be used to track active versions of the proteases by SDS-PAGE, or to affinity purify the desired protease. The authors identified first-generation biotin-labeled peptides that retained potency and were highly specific for the target protease. To demonstrate specificity, enzymes incubated with each ABP were run on a gel, transferred to a membrane, and detected with fluorescently labeled streptavidin on the Sapphire.

The components and pathways that make up the classical blood coagulation cascade. Dr Graham Beards. Licensed under CC BY-SA 3.0
The components and pathways that make up the classical blood coagulation cascade. Dr Graham Beards. Licensed under CC BY-SA 3.0

The blood coagulation pathway

Blood coagulation involves a proteolytic signaling pathway in which a series of zymogens are activated by peptide bond cleavage to become proteases that in turn cleave additional targets. The pathway culminates in the cleavage of prothrombin to create thrombin, which promotes clot formation by activating platelets and creating fibrin from fibrinogen. Disruption of the blood coagulation pathway or abnormal activity of its players is associated with disorders ranging from hemophilia to cancer to Alzheimer’s disease. Therefore, understanding the pathway and how it is regulated is important to a variety of lines of research. 

Challenges to studying regulation of the blood coagulation pathway

Activated protein C (APC), thrombin, and factor Xa, are proteases important to regulating the blood coagulation pathway and maintaining the correct balance of coagulation and blood flow. However, all three are vitamin K–dependent serine proteases and have similar structures and substrate preferences. The structural and substrate similarities have made it difficult to tease apart the roles of each of these three proteases in biological systems and disease states.

In addition to multicolor fluorescent imaging of blots and gels, the Sapphire Biomolecular Imager can carry out chemiluminescence, densitometry, white light, phosphor, and near infrared imaging of blots, gels, tissues, microwell plates, and more. Learn more about the Sapphire Biomolecular Imager and how Azure can support your research by clicking here.

 

How is PCR used in genotyping?

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qPCR

The polymerase chain reaction (PCR) is used in many genotyping approaches and methodologies. Just simple PCR can be used for genotyping in situations where a known genetic sequence is being tracked, such as identifying model organisms carrying a transgene. PCR primers can be designed to only amplify a product when the animal’s DNA is carrying the specific sequence scientists have introduced. In these cases, the expected PCR product will be identified as a band on a gel.

Allele-specific PCR

Allele-specific PCR involves designing PCR primers that amplify different products depending on the genotype of the sample. Allele-specific PCR strategies can identify whether a sample is heterozygous or homozygous for a specific genetic variant based on whether one or two products are produced. Allele-specific PCR reactions can be analyzed by examining the sizes of the PCR end products on a gel or can be analyzed by following the accumulation of fluorescence using real time PCR (RT-PCR).

Using RT-PCR to detect SNPS

RT-PCR is frequently used to detect SNPs. With careful planning, PCR primers can be designed that are sensitive to the single base change at the location of the SNP and will only anneal to one variant so that a PCR product is only produced when the variant matches the primer exactly. In this type of system an intercalating fluorescent dye such as SYBR green can be included in the PCR reaction to follow the accumulation of the PCR product in real time. Other strategies incorporating fluorescently tagged primers or probes have been developed that increase the specificity of the approach and allow multiplexing, the detection of two or more products in the same PCR reaction by real-time PCR.1

PCR in genotyping
Figure 1. View of the Cielo's Experiment Area screen showing the increase over time of the fluorescent signals reflecting PCR product accumulation in a multiplex real-time PCR reaction

Other ways to detect SNPS

Different PCR strategies can also be incorporated into genotyping approaches that can be used even if you don’t know anything about the genome of the organism you are studying. In random amplified polymorphic DNA (RAPD), random 10-mer primers are used to carry out PCR, and the products generated across different samples are compared, usually by separating them on a gel and comparing the sizes of the amplified products. For the approach to work, the combination of primers must produce products and in some fraction on individuals, genetic variation changes the size or number of PCR products. With luck, some of the genetic differences reflected in the different PCR products will associate with the phenotype of interest.2

Using amplified fragment length polymorphism to genotype

Another genotyping strategy that incorporates PCR and does not require knowing the sequence of the genome of the organism being studied is amplified fragment length polymorphism (AFLP). In AFLP, the genomic DNA is digested with restriction enzymes, adapters are ligated to the digested ends, PCR is carried out using primers directed towards the adapters, and the products are separated and analyzed by gel electrophoresis. As in RAPD, the method is useful if there are some differences in PCR products that are associated with the phenotype of interest.3

SOURCES

  1. Broccanello C et al. Comparison of three PCR-based assays for SNP genotyping in plants. Plant Methods. 2018;14:28.

  2. Random Amplified Polymorphic DNA (RAPD) at the National Library of Medicine website. https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/. Accessed October 31, 2022.

  3. Paun O, Schönswetter P. Amplified Fragment Length Polymorphism (AFLP) – an invaluable fingerprinting technique for genomic, transcriptomic and epigenetic studies. Methods Mol Biol. 2012; 862:75-87.

Sapphire Biomolecular Imager used in investigation of potential nasal vaccine for COVID-19

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COVID-19 Imaging

Vaccines have been instrumental in the public health response to the SARS-CoV-2 pandemic. The existing approved vaccines induce neutralizing antibodies and are very effective at preventing symptomatic infection. However, current vaccines do not elicit sterilizing and mucosal immunity necessary to avoid breakthrough infections.

Testing an attenuated version of SARS-CoV-2

In a recent publication, Ye et al (2) set out to make an attenuated version of SARS-CoV-2 and test its utility as a vaccine. The authors created a recombinant strain with a point mutation that inactivates NSP16, a protein needed to cap viral RNAs and prevent them from being detected by the immune system. The mutation, D130A, was known to inactive the analogous enzyme in other coronaviruses. The mutant SARS-CoV-2 strain was characterized to determine if (1) the virus was viable, (2) the ability of the virus to replicate was attenuated, and (3) the virus could be a candidate for vaccine development.

Fig. S1. Genetic stability of SARS-CoV-2 d16. A Detection of d16 gene during viral passage in VeroE6 cells. RNAs were extracted from the d16-infected cells of P0 to P10 passages. RT-PCR was performed with a primer pair flanking the d16 mutation. The 297-bp PCR products were resolved by agarose gel electrophoresis (arrowhead). The passage numbers were denoted at the top of each lane. B Sanger sequencing.

The findings

The answer to all three questions was “yes”. The virus was viable in in vitro assays, but its growth was attenuated both in vitro and in vivo. Hamsters infected with the mutant virus had significantly lower viral loads in the upper and lower respiratory tracts than hamsters infected with the wildtype virus, and infection with the attenuated virus did not kill transgenic mice as wildtype virus did.

Use of the mutant strain as a live vaccine was tested in hamsters using a single dose regimen. Hamsters were inoculated intranasally with wildtype or mutant virus, monitored for 28 days, then challenged with wildtype virus on day 29. On day 28, the presence of neutralizing antibodies was assessed using a live virus neutralization assay that detected bound virus using a fluorescently-labeled antibody. The fluorescent signal was assessed and quantified using the Azure Sapphire Biomolecular Imager.

Exposure to the mutant strain was found to be as effective as the wildtype virus at preventing SARS-CoV-2 infection. Viral titers in the lung and respiratory tract were so low the vaccination may have elicited sterilizing immunity. Indeed, hamsters co-housed with infected but vaccinated hamsters never became infected themselves. Promisingly, spike protein-specific IgA was detected in the serum and bronchoalveolar lavage fluid of vaccinated mice, suggesting mucosal immunity was stimulated.

Ye et al conclude the results provide strong justification to pursue further developing the strain as a potential vaccine.

This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo credit: Alissa Eckert, MSMI; Dan Higgins, MAMS.
This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo credit: Alissa Eckert, MSMI; Dan Higgins, MAMS.

How COVID infections begin

COVID infections usually begin in the upper respiratory tract mucosa. Therefore, stimulating mucosal immunity may be most effective at preventing infection. Mucosal immunity is independent of the systemic immune system and is mediated primarily by secretory IgA antibodies. Injectable vaccines such as those used for flu and pneumonia primarily induce serum IgG, which may protect the lower respiratory tract and prevent systemic spread. Inducing upper airway mucosal protection may require nasal immunization. Additionally, in some experiments inducing a mucosal cellular immune response has required immunizing with whole, wild-type or attenuated viruses or bacteria.(1)

Live attenuated vaccines use a weakened form of the infectious agent (virus or bacterium). The agent can replicate enough to stimulate a strong immune response but not so much as to cause disease. Live attenuated vaccines result in a strong and long-lasting immune response, are used to protect against several agents, and were crucial to the eradication of smallpox and the near eradication of poliovirus.

In addition to fluorescent imaging of tissue culture plates, the Sapphire provides densitometry, phosphor, multichannel fluorescence, near-infrared, chemiluminescence, 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

SOURCES

  1. Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nature Med. 2005;11:S45-S53.
  2. Ye ZW, Ong CP, Tang K, et al. Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals. Cell Mol Immunol. 2022;Mar 29;1-14.
  3.  

In-cell Westerns used to Demonstrate Potent Anti-inflammatory Activity of Mosquito Saliva Protein

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Imaging In-cell Western

Both the Azure 300 and Azure Sapphire Biomolecular Imager, along with multiple reagents from Azure Biosystems, were used in a recent publication revealing anti-inflammatory activity of a mosquito saliva protein. Shrivastava et al from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (NIH) in Rockville, MD, employed both the Azure 300 and Sapphire, as well as fluorescent blocking buffer and secondary antibodies from Azaure, in their study demonstrating the protein Alboserpin inhibits pro-inflammatory activities of the coagulation cascade protein factor Xa (FXa).

Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo
Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. Licensed under CC BY 4.0. In-Cell Western blots conducted using Azure fluorescent secondary antibodies and imaged on the Azure Sapphire Biomolecular Imager demonstrate a decrease in Fxa-induced phosphorylation of EKR 1/2 in the presence of Alboserpin.

Research Applications with the Azure 300 and Azure Sapphire Biomolecular Imager

By traditional and in-cell Western blots, Alboserpin was shown to block the FXa-induced increase in phosphorylated ERK1/2, indicating it blocks FXa-induced ERK1/2 signaling. The chemiluminescent blots were imaged using the Azure 300, while the in-cell Westerns were imaged on the Sapphire with fluorescent blocking buffer and fluorescent secondary antibodies.

These results from the NIH provide additional insight into the mechanisms mosquito vectors employ to avoid host responses during blood feeding. The authors indicate further research is needed to examine the role Alboserpin might play in pathogen transmission.

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Alboserpin and its role on FXa

FXa is known to trigger inflammation by signaling through protease-activated receptors (PARs). In their recently published work, Shrivastava et al demonstrate that Alboserpin has anti-inflammatory activity, in vitro and in vivo, due to its inhibition of FXa activities. Across multiple experiments, Alboserpin was shown to prevent numerous FXa-induced processes including FXa-induced release of proinflammatory cytokines, increased expression of multiple PARs and other proteins (specifically PAR-1, PAR-2, PAR-3, NF-kB, ICAM, and VCAM-1), induced activation of NF-kB, and cleavage of PAR-2.

What are Aedes mosquitos? Why is their saliva important?

Aedes mosquitos are an important vector for arthropod-borne (arbo) viruses such as Zika, dengue, and chikungunya viruses. Arbo viruses are responsible for substantial disease burden, morbidity, and mortality among human populations world-wide. These viruses can be introduced into a host when the mosquito pierces the skin to draw blood and releases saliva at the site of the bite.

The mosquito saliva contains proteins that prevent hemostasis (a process that involves reduced blood flow), coagulation, and clot formation to prevent blood loss at the site of an injury. The Aedes albopictus mosquito has an anticoagulant called Alboserpin in its saliva. Alboserpin is a serine protease inhibitor known to demonstrate competitive, reversible, and high-affinity binding to FXa, an important component of the coagulation cascade.

More about in-cell Westerns: History behind In-cell Westerns

In addition to chemiluminescent and multicolor fluorescent imaging of blots and multi-well plates, the Sapphire Biomolecular Imager can carry out densitometry, white light, phosphor, and near infrared imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

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SOURCE
  1. Shrivastava G, Valenzuela-Leon PC, Chagas AC, et al. Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. ImmunoHorizons. 20022;6(6):373-383.

Azure Biosystems Awarded SelectScience Seal of Quality Award

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Press Releases

Dublin, Calif. – August 5, 2022 – Azure Biosystems has earned its first SelectScience® Seal of Quality award, in recognition of outstanding feedback received from scientists globally.

The Azure cSeries Gel Imaging Systems have been awarded a Bronze Seal of Quality in recognition of having received over 50 reviews from scientists globally, with an impressive overall rating of 4.9 out of 5 stars.

bronze seal of quality

“I would like to take this opportunity to thank all our reviewers around the world for sharing their valuable opinions and also to congratulate Azure Biosystems, who have been recognized by the people who matter most, their customers,”

Kerry Parker, SelectScience CEO

The Azure Imaging Systems (which have replaced the Azure cSeries Imaging Systems) provide fast, flexible imaging for Western blotsgels, and more. Systems are available with multiple imaging modes including chemiluminescence, visible light, fluorescence and laser-based near infrared imaging. Compared to the original cSeries, the Azure Imaging Systems include a new 9 megapixel camera for even higher resolution and faster imaging and offer four-channel detection for multicolor and quantitative fluorescent Western blotting. Visit Azure Biosystems to learn more about the Azure Imaging Series.

Seals of Quality recognize the top 0.1% of products that consistently get the highest customer review ratings on SelectScience®, a leading independent laboratory technology website, and are designed to help scientists see, at a glance, the instruments and services their peers love the most.

There are four levels of Seal of Quality: Platinum, Gold, Silver, and Bronze, differentiated by how many reviews a product has received and its overall average rating. This marks the first Seal of Quality to be awarded to Azure Biosystems. 

Read more reviews on Azure instruments by clicking here. For more about the latest Seal of Quality winners, see the SelectScience website.

For additional information, please visit https://azurebiosystems.com.

Contact

Lisa Isailovic, VP of Marketing, Azure Biosystems
lisa.isailovic@azurebiosystems.com
(925) 307-7127

Phosphor Imaging with the Azure Sapphire in an Investigation of Chromosome Segregation

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Imaging

New insight into microtubule-kinetochore interactions

Work recently published in the Journal of Cell Biology1 provides new insight into a fundamental processes of cell biology, chromosome segregation. Flores et al investigated the importance of three specific points of interaction between Dam1 and Ndc80 to the strength and persistence of attachment between the kinetochore and microtubule tips.

This research group previously identified the three regions of interaction. Most recently, they mutated or phosphorylated amino acids in each of the three regions and used laser trapping to assess the effect of the mutations on the persistence of the interaction under constant force and on the rupture strength of the interaction when the applied force was rapidly increased.

Autoradiograph of an SDS-PAGE gel using phosphorimaging from Azure Sapphire
S3. Analysis of phosphorylation levels on wild-type and mutant Dam1 complexes. (A) Autoradiograph of an SDS-PAGE gel (8–14%) showing incorporation of [γ-32P]ATP into different interaction regions of the Dam1 complex. (B) Coomassie blue–stained SDS-PAGE gel (8–14%) of wild-type Dam1 complex with GST-Sli15554–698 and GST-Ipl1p (wild-type), GST-Sli15554–698 alone, GST-Ipl1p alone, and GST-Sli15554–698 together with GST-Ipl1p. Licensed under CC BY 4.0.

Phosphorylation using radioactive 32P-ATP

To measure the extent of phosphorylation of Dam1 proteins, the authors carried out a phosphorylation assay using radioactive 32P-ATP. Radio-labeled products were detected on dried SDS-PAGE gels by phosphor imaging on an Azure Sapphire Biomolecular Imager (Figure S3-A.)

The results of the experiments showed that all three regions contribute to the strength and persistence of attachments between the microtubule tips and kinetochores. Two of the regions also contributed to the rupture strength of the interaction. However, phosphorylation of one site had no effect on rupture strength but decreased attachment lifetime. The authors hypothesize these differential effects on long-term stability vs short-term strength could play a role in error correction in vivo.

What happens when a cell divides

Phases of Mitosis
Phases of Mitosis

When a cell divides, it is essential that each daughter cell receives a complete set of chromosomes. During mitosis, chromosomes condense and become attached to the ends of microtubules that make up the mitotic spindle. As the cell prepares to divide, the spindle microtubules shorten and drag the attached chromosomes to opposite ends of the dividing cell.

Incorrect chromosome segregation is usually lethal since the daughter cells will not receive a complete set of genomic information. In multicellular organisms, chromosome mis-segregation is associated with cancer.

How exactly the chromosomes attach to the spindle microtubules is unknown. The tips of the microtubules attach to a large multi-protein structure attached to each chromosome called the kinetochore. However, the microtubules change length by adding or removing tubulin subunits to the tips so important questions remain including how a chromosome attaches to a dynamic structure, as well as how a chromosome remains attached to a microtubule under force while being pulled towards the spindle pole.

How do kinetochores attach to the microtubule tips?

A protein complex called Ndc80 is required to keep chromosomes attached to the spindle. Experiments in yeast have demonstrated that all four subunits of Ndc80 are essential. In yeast, Ndc80 interacts with another multiprotein complex, Dam1. Dam1 appears to form rings around microtubules and to help the kinetochore-spindle attachment bear load and remain attached while the microtubule changes length.

How do kinetochores attach to the microtubule tips?

The Aurora B kinase is required for accurate chromosome segregation. Phosphorylation of targets by this kinase causes kinetochore-microtubule attachments to release. It is hypothesized that incorrect attachments are not under tension and that the kinase recognizes these non-functional attachments and phosphorylates target sites to break the attachment.

In addition to phosphor imaging, the Sapphire Biomolecular Imager provides densitometry, multichannel fluorescence, near-infrared, chemiluminescence, 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.
SOURCE
  1. Flores RL, Peterson ZE, Zelter A, et al. Three interacting regions of the Ndc80 and Dam1 complexes support microtubule tip-coupling under load. J Cell Biol. 2022;221(5):e202107016

How a Type 2 Diabetes Treatment Reduces Cardiovascular Risk

Categories
2D DIGE Fluorescence imaging

In recent work, Ekhzaimy et al1 took advantage of the three-channel fluorescence imaging capability of the Sapphire Biomolecular Imager to conduct a proteomics study of liraglutide treatment for type 2 diabetes.  

2D DIGE to compare baseline and post-treatment patient samples

In this study, two-dimensional difference gel electrophoresis (2D DIGE) was used to compare the plasma proteome of 20 patients before and three months after liraglutide treatment. Baseline and post-treatment samples from each patient were labeled with Cy3 or Cy5 using a dye-switching strategy to control for any dye-specific bias. To control for gel-to-gel variation, equal amounts of all samples were pooled and labeled with Cy2 as an internal control. For a given patient, the two labeled samples were combined with the internal control and run on a 2D gel which was scanned for Cy2, Cy3 and Cy5 using the Sapphire Imager. Overlaying the images and quantifying spot intensity identified 128 spots whose intensity was statistically significantly different after liraglutide treatment.

Example of a 2D DIGE experiment in which one sample was labeled with a red dye and the other with green.
Example of a 2D DIGE experiment in which one sample was labeled with a red dye and the other with green. Spots in yellow are found in both samples and result from the superposition of the red and green images. Image available here and licensed under the Creative Commons Attribution-Share Alike 2.0 Generic license.

To identify the proteins whose expression was affected by liraglutide treatment, a preparative gel was run with a pool of equal amounts of each patient protein sample. The spots of interest were cut from the preparative gel and the protein identified by mass spectrometry. Of the 128 spots, 72 were identified and represented 29 unique proteins. The identities of two spots were further confirmed by chemiluminescent Western blot, also imaged using the Sapphire. 

Results from the study

The results indicate that liraglutide affects expression of proteins involved in inflammation, apolipoprotein regulation, and oxidative stress, all processes known to contribute to cardiovascular disease. The authors conclude that effects on these pathways may contribute to the effectiveness of liraglutide treatment and that the results should be further investigated in larger-scale studies involving more patients and longer terms of treatment. If substantiated, the authors suggest the results could lead to the development of biomarkers to assess the cardiovascular benefits of liraglutide. 

Using an untargeted proteomic approach

Liraglutide, a glucagon-like peptide-1 receptor agonist, improves glycemic control through action on the pancreas. Liraglutide also reduces cardiovascular risk in patients with diabetes via an unknown mechanism. The authors used untargeted proteomic profiling of plasma from patients treated with liraglutide to identify changes induced by treatment and to see if those changes in the proteome provide insight into liraglutide’s cardiovascular and metabolic benefits.  

An advantage of the untargeted proteomic approach used in this research is the potential to discover unexpected things. With an untargeted approach, any protein whose level changes as a result of treatment may be identified, whereas in targeted experiments, only specific candidate proteins are studied to prove or disprove their involvement in a process. Metabolism is complex and involves many interacting pathways and processes, making it an attractive subject for an untargeted approach that can assess many targets simultaneously. In addition, protein levels may better reflect changes in physiology than RNA levels or DNA-level changes. 

In addition to multi-color fluorescence and chemiluminescence imaging, the Sapphire Biomolecular Imager provides densitometry, phosphor, 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.

SOURCE
  1. Ekhzaimy AA, Masood A, Benabdelkamel H, et al. Plasma proteomics reveals an improved cardio-metabolic profile in patients with type 2 diabetes post-liraglutide treatment. Diab Vasc Dis Res. 2022;19(3):14791641221094322.

Western Blotting Reagents Roundup – July 2022

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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. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

Phosphorylated MED1 links transcription recycling and cancer growth

Aberrant transcription goes hand-in-hand with oncogenesis. Chen et al1 used Chemi Blot Blocking Buffer and Radiance ECL in Western blot experiments investigating transcription recycling in cancer cells. Uncontrolled transcription initiation and elongation are known to be associated with tumor growth but the authors examined whether Pol II recycling, in which RNA polymerase II re-transcribes the same gene rather than being released after transcription is complete, is also associated with cancer. Using a recycling assay developed in their prior publications, the authors demonstrated that Mediator 1 (MED1), when phosphorylated by CDK9, drives Pol II recycling.

Phosphorylation of MED1 increased during prostate cancer progression and inhibiting CDK9 decreased MED1 phosphorylation, Pol II recycling, and prostate tumor growth. The results suggest MED1 phosphorylation and transcription recycling are involved in cancer growth, and MED1 phosphorylation may provide a biomarker to assess therapeutic response of cancers to CDK9 inhibitors.

SHOP: Chemi Blot Blocking BufferRadiance ECL

Aripiprazole Offsets Mutant ATXN3-Induced Motor Dysfunction

Machado-Joseph disease (MJD) is a dominantly inherited progressive ataxia caused by expansion of a CAG repeat in the ataxin-3 gene. Jalles et al2 used Radiance ECL and AzureRed total protein stain, in addition to the Sapphire Biomolecular Imager, in a study investigating how the antipsychotic drug aripiprazole suppresses MJD pathogenesis. In a C elegans model of MJD, the authors found that aripiprazole improved motor performance and this improvement depended on dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors. Identifying the specific targets of aripiprazole may help develop new therapeutics for MJD with fewer side effects.

DISCOVER: Sapphire Biomolecular Imager

SHOP: Radiance ECLAzureRed

MARK2 regulates directed cell migration

During metastasis, cancer cells migrate by building out the cytoskeleton at the leading edge of the cell and retracting it at the rear. Pasapera et al3 used Azure’s Radiance ECL in a study of cancer cell cytoskeleton polarization. The authors investigated whether the kinase MARK2, known to regulate the microtubule cytoskeleton in other processes, plays a role in the polarization of the cytoskeleton and directed migration of cancer cells. In osteosarcoma cells, Western blot experiments demonstrated that MARK2 promotes stress fiber formation and myosin II activation and mediates inactivation of myosin phosphatase.

The data suggests MARK2 is a major regulator of cell contractility and adhesion that mediates cancer cell motility.

SHOP: Radiance ECL

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli

Alport syndrome is a congenital, progressive glomerular disease that leads to the progressive loss of kidney function. Madison et alused Radiance ECL and TotalStain Q as well as an Azure 600 Imaging System in a study characterizing the glomerular basement membrane (GBM) in a mouse model of Alport syndrome. The investigators found that collagen a1(III) was deposited in the GBM of Alport mice; in wild type mice, collagen a1(III) is found only in the mesangium.

Quantitative Western blotting was carried out using total protein normalization with TotalStain Q staining as the control and the quantitative Westerns confirmed increased levels of collagen a1(III) in the glomeruli of Alport mice. The presence of collagen a1(III) was found to activate DDR1 receptors and lead to changes in gene expression consistent with podocyte injury. Lack of either of the two collagen receptors on podocytes has previously been shown to slow disease progression. The results indicate aberrant collagen-mediated co-receptor signaling through the DDR1 and a2b1 integrin receptors contribute to podocyte injury and renal pathology in Alport syndrome.

DISCOVER: Azure 600 Imaging System

SHOP: Radiance ECLTotalStain Q

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 on the left.

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SOURCES

  1. Chen Z, Ye Z, Soccio RE, et al. Phosphorylated MED1 links transcription recycling and cancer growth. Nuc Acids Res. 2022;500(8):4450-4463.
  2. Jalles A, Vieira C, Pereira-Sousa J, et al. Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in elegans. Biomedicines. 2022;10(2):370.
  3. Pasapera AM, Heissler SM, Eto M, et al. MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Curr Biol. 2022;32(12):2704-2718.
  4. Madison J, Wilhelm K, Meehan DT, et al. Glomerular basement membrane deposition of collagen a1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin a2b J Pathol. 2022; doi: 10.1002/path.5969.

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

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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.

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.

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.

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.

Why is there amplification in the no-template control?

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.

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