Western Blotting Reagents Roundup – June 2024

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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.

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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.

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