Western Blotting Reagents Roundup – June 2024

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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.

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

Featured Studies in this Reagent Roundup

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SHOP: AzureRed

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

Previous Reagent Roundups:

Read other blog posts about publications using Azure:

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SOURCES

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

Which Imager is Best for Total Protein Normalization?

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Fluorescence imaging Imaging Western Blotting
Total protein normalization (TPN) is used to quantify the abundance of the protein of interest, without having to rely on housekeeping genes. It is usually done by incubating the membrane with a total protein stain. 
TPN has become the preferred method for normalizing Western blot data. But to fully leverage its benefits, you need an imaging system that allows you to multiplex TPN with detection of your target proteins. Fortunately, we have just what you need! Whether you want to combine TPN with near infrared (NIR) fluorescent Western blotting, or with enhanced chemiluminescent (ECL) detection, read on to learn how these Imaging Systems from Azure enable you to do so with ease.

Why you should use Total Protein Normalization (TPN)

A major advantage of TPN is that it delivers more accurate quantification of target analytes than the established practice of using individual housekeeping proteins. This is because TPN is less susceptible to change in response to experimental treatments, providing a more reliable baseline against which target protein expression can be compared. TPN also avoids the problem of over-saturation where low abundance analytes require high protein loads to reach the necessary sensitivity for detection since it has an incredibly broad dynamic range (1–50 μg of lysate).

HeLa-lysate-5μg-replicates-imaged-using-TotalStain-Q
HeLa lysate 10μg replicates imaged using TotalStain Q (Cy3 channel, green) and pSTAT3 on an Azure Sapphire Imager.

How is TPN currently performed?

Current methods for TPN vary according to the chosen readout. Where TPN is combined with ECL detection, it is common practice to use specialized gels that chemically modify all proteins within each sample upon exposure of the gel to UV light, enabling their subsequent measurement. A drawback of this approach is that it has only a narrow range in which the protein load is linear.

In situations where TPN and NIR detection are paired, two distinct techniques are used. The first involves labeling the entire protein population of each sample with a fluorescent dye before loading the gel, a process that introduces an additional source of variability to the workflow. The second requires that the membrane be stained with a NIR reagent for TPN immediately after transfer. The membrane is then imaged and de-stained prior to NIR target detection. Because de-staining is never 100% complete, this latter method essentially restricts target detection to just one of the two available NIR channels.

How does Azure's approach to TPN improve on existing methods?

Azure’s range of imaging systems includes several models that allow target protein detection to be multiplexed with TPN– without the need for dedicated precast gels, laborious stripping efforts, or re-probing to be done.

TotalStain Q PVDF for Western blotting from Azure Biosystems
TotalStain Q is an accurate and quantitative fluorescent total protein stain to perform normalization of Western blots imaged on both camera-based and laser-based instruments with green channel or Safe-Dyes channel.

For the best TPN results, treat your blots with a total protein stain like TotalStain Q between protein transfer and blocking, and process them as you would normally. TotalStain Q has a broad linear range, which makes it ideal for total protein normalization of low expressed proteins that require up to 50μg lysate/lane. Always make sure the total protein stain you use is compatible with the antibody binding and detection method.

Which imagers support TPN?

By reserving the NIR channels for your proteins of interest, sensitivity is uncompromised by integrating TPN into your Western blotting workflow. For TPN with NIR Western blot detection, using imaging systems such as the Azure 500Q, Azure 600, and the new Sapphire FL will provide detection of target analytes in the 700nm and 800nm channels. They also include a third channel used to measure TotalStain Q.

Azure Biosystems Sapphire FL for total protein normalization
The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend the standard 532 optical module for detection of TotalStain Q.

The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend using the standard 532 optical module for detection of TotalStain Q. Select Azure imaging systems also enable target protein detection to be multiplexed with TPN. The Azure 300Q and Azure 500Q both include the Q module (our optional green fluorescence channel) to quantify total protein staining. 

Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers
Shown: Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers. You are able to add a Q Module for Total Protein Normalization of NIR Western blots using TotalStain Q. The upgrade Azure 500Q will also detect one more fluorescent target.

In addition, the Azure 600 imager is also able to image both chemiluminescent and fluorescent signals. It comes readily equipped the 524nm laser to be able to detect TotalStain Q as well.

The Azure 600 Imager is a fully equipped laser scanning system capable of fluorescent, NIR, chemiluminescent, blue light, and UV imaging.

Where TPN and ECL are performed in parallel, the Azure 300Q is a compact benchtop solution that can readily be upgraded to include visible and/or NIR fluorescent detection capabilities as your Western blotting requirements evolve.

Azure 300Q imager with green channel for total protein normalization
Add visible and/or NIR fluorescent detection capabilities as your needs evolve with the Q Module. The Azure 300Q imager is equipped with a green channel for total protein normalization.

And if you already have an Azure 300 or Azure 500 in your lab, adding our optional green fluorescence channel – the Q module – to your system means you can easily begin multiplexing TPN without interruption to your Western blotting workflow. It’s easy to do so, just contact us to upgrade your imager.

Want to find out how you can add multiplex total protein normalization with NIR fluorescent Western blot detection or ECL to your research? Send us a message using the form on this page. 

BONUS: We’re giving away free samples of TotalStain Q – our newest reagent for total protein staining! Grab a sample!

If you’d like to learn more about how TPN can enhance your Western blotting data, check out this webinar:

Western Blotting Reagents Roundup – August 2023

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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. This quarter, we’re highlighting four recent publications that used Azure reagents to achieve excellent Western blotting results.

Featured Studies in this Reagent Roundup

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Previous Reagent Roundups:

Read other blog posts about publications using Azure:

Shop Reagents Mentioned

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

SOURCES

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

Multiplex Western Blots: 6 Strategies that Work

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Multiplex Western Blotting

Are you having problems with your multiplex fluorescent Western blots? In this post, we’ll cover the exact protocols in more detail in some of our application notes that cover phosphorylation/total protein detection, and single step normalization.

Detecting one protein, two proteins, three proteins? How about FOUR proteins on the same Western Blot? Multiplexing your Western blots allows you to save money on each experiment.

Here at Azure Biosystems we’re great believers in the power of fluorescent multiplexing in Western blots, which has driven a lot of the development of our line of Azure imagers. But we recognize that taking the step from a standard HRP/chemiluminescent-based approach can be daunting. Keep reading for 6 tips to make the switch as easy as possible.

Azure fluorescence capabilities vs. competitor fluorescence, 3 proteins, plus total protein staining, and multiplex fluorescent Western blot

Browse our 6 multiplex Western blot expert tips

AZURE EXPERT TIP #1: Increase your concentrations

The concentration of both the primary and secondary antibody required may be increased compared to chemiluminescent Westerns depending which imager you’re using. In more modern or laser-based imagers, this effect may be less marked.

AZURE EXPERT TIP #2: Test individually first

Going hand in hand with our first tip, it always makes sense to test your antibodies individually first. That way you can determine optimal concentrations, factors contributing to background or lack of specificity in a simpler environment, rather than try to unpick 3 different primary and secondary antibodies at once.

AZURE EXPERT TIP #3: Use adsorbed secondary antibodies

Although it sounds simple, many people don’t consider that they are now adding multiple antibodies from multiple species onto a single blot. If you work with multiple fluorescence in other fields you’ll already be aware of the importance of cross-adsorbing secondary antibodies to reduce inter-species cross reactivity. But many people don’t consider this for Westerns, it’s worth checking out your antibodies to ensure they meet the grade.

Multiplex fluorescent Western blot
Multiplex fluorescent Western blot imaged with an Azure Imager using Cy3 and Cy5.

AZURE EXPERT TIP #4: Expand the spectrum with multiplexing

Three-color Western blots are exciting, but what about five colors? With near-infrared capability, the number of spectrally distinct peaks that can be isolated can be increased greatly. Obviously, this may require a bit of work up to get the antibodies optimized, but imagine the time and cost savings generated by performing one Western for five proteins.

The Sapphire Biomolecular Imager is an imager capable of detecting up to four proteins on the same Western blot, which allows you to save money on reagents each time you do an experiment. It uses 4 fluorescence channels to easily quantify overlapping bands. Read more on multiplex protein detection here

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

AZURE EXPERT TIP #5: Check your membrane

Some membranes will auto-fluoresce when exposed to UV light generating a high background signal, although more and more fluorescent safe membranes are being developed. We would also recommend switching to using PVDF membranes from nitrocellulose due to its increased sensitivity, as we discussed previously.

Package of pre-cut PVDF membranes for Western blotting applications
PVDF membranes reduce background noise for improved sensitivity.

AZURE EXPERT TIP #6: Choose the right channel for your protein

Unfortunately all detection channels were not created equally. For standard fluorescence use blue to detect your highest abundance protein, green the middle and red for your lowest abundance protein. If introducing NIR the excellent sensitivity and low background achieved with these fluorophores also makes them ideal for low abundance proteins.

Want to read more? Check out this paper from the National Institute of Health that utilizes multiplex Western blots using microchip electrophoresis.

Have more questions or want to learn more about multiplexing and how it can improve the way you research? Fill out the form on the left- we’ll be in touch.

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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.

Featured Studies in this Reagent Roundup

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

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.

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.

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.

Lipid peroxidation in sporadic Alzheimer's disease

Western blot imaged by fluorescence immunoblotting using Azure Sapphire Imager

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 Imager (Figure 3C and Figure 3D).

The 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.

DISCOVER: Azure Sapphire Biomolecular Imager

TRY BLOCKING BUFFER: Free fluorescent blocking buffer samples

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 

DISCOVER: Azure 300 Imager

TRY RADIANCE ECL: Free Radiance, Radiance Q, and Radiance Plus Samples

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

Previous Reagent Roundups:

Read other blog posts about publications using Azure:

Shop Reagents Mentioned

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

SOURCES

  1. 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.

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.

Featured Studies in this Reagent Roundup

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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New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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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.

Handy Resources to Improve Western Blots

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Troubleshooting Western Blotting

At Azure Biosystems we live and breathe all things Western blotting. From innovative imagers, to top notch reagents and accessories, to customer education, we are here to support you in your quest for the most robust Western blotting protocols, techniques and data no matter where you are in your Western blotting career.

Are you a Western blotting novice trying to learn more about the process to perfect your technique? Or maybe you’re a veteran Western blotter looking to improve your blots or switch detection methods. In either case, bookmark this page to use as a list of Western blotting resources and troubleshooting. Check out the handy infographic below to see which reagents you may need to get started on your Western blot.

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

Helpful resources to IMPROVE your Western blotting game:

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WATCH: Troubleshooting Common Issues with Western Blotting

Potential Treatment for Advanced Kidney Cancer

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Protein Assays Publication Spotlight 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.

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We’d love to read it! Email your publication to us and we’ll send you something for sharing.

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

Help! Why do my Western blots look terrible?

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Western Blotting

One of the most common questions when troubleshooting problematic Western blots is, “Why is the background so high?”

High or uneven background doesn’t only look bad- it also 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 Background in Western Blots

STEP 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.

STEP 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.

Unsure which blocking buffer to use? Click below for a free sample.

STEP 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 the blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer.

STEP 4: Find the best exposure time for your chosen detection method

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 using an imager like the Azure 600, or another digital imager with a CCD camera makes capturing multiple exposure times even easier.

Azure 600 Western blot Imaging system
The Azure 600 is the only system that offers two channel, laser-based IR and chemiluminescent detection, with the speed and sensitivity of film, with the ability to image visible fluorescent dyes, standard EtBr and protein gels, and infrared laser excitation for quantitative Western blot imaging in the NIR. This catchall Western blot imager improves your data quality imaging with infrared dyes and offers signal stability.

If you’re using an ECL detection system, use Radiance ECL, a detection reagent with a stable, long-lasting signal, so exposure times are predictable and reproducible. Using Radiance helps ensure the signal doesn’t decay so rapidly that you cannot conduct multiple exposures.

STEP 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.

Quick Tips to Keep in Mind for Fluorescent Western Blots

  • Azure Quick Tip #1: Change your membrane

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

  • Azure Quick Tip #2: Remember that wet membranes can also autofluorescence

    Dry the membrane completely before imaging.

  • Azure Quick Tip #3: Control the temperature during the protein transfer step

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

With these tips, you’re on your way to reducing the background and getting clean, clear Western blots. If you still have questions, fill out the form on the right and one of our experts will reach out to assist.

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Regulation of Gene Expression by Enhancer RNAs

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Imaging Publication Spotlight 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.

More research done with the Azure Sapphire: