Duolink® Webinars

 Introduction to Duolink and PLA Technology – March 10, 2015

For researchers studying biological pathways who require improved sensitivity to uncover disease biomarkers, Duolink using PLA technology, when combined with qualified antibodies, outperforms traditional immunoassays, such as co-IP, through a simple and sensitive in situ detection of endogenous proteins.

This webinar features an introduction to the Duolink technology and applications of PLA and it’s many variants.

Duolink: A Versatile Tool for Detection, Quantification and Cellular Localization of Protein Modifications or Complex Formations
Presented by Dr. Tom Juehne, Sigma-Aldrich

Visualization of protein-protein interactions at endogenous levels of expression is a powerful advancement in Life Science. Duolink using PLA technology allows direct visualization of cell signaling pathways providing insights into phenotypic responses of healthy and disease states. Duolink is a versatile tool for detection, quantification and localization of cytoplasmic signaling events. PLA provides the ability to see and quantitate protein posttranslational modifications, define complex interaction, illuminate complex protein clusters or amplify low abundant single events. Duolink has also been used to demonstrate the ability to visualize translocation events. We will discuss and show the versatility of Duolink using PLA technology and demonstrate this powerful tool for use in the study of Life Science at the endogenous cellular level.

Introduction to PLA and Duolink
Variants of the Proximity Ligation Assay Technology

Presented by Dr. Ola Söderberg, Uppsala University

The activity status of a protein or signaling networks can be visualized with in situ Proximity Ligation Assays (in situ PLA) using a pair of antibodies equipped with DNA oligonucleotides (proximity probes) to target interacting proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which is a surrogate marker for the interaction. I will describe how different versions of in situ PLA may be used to also analyze protein interactions, post-translational modifications and protein-DNA interactions. I will also describe a multiplexed version of in situ PLA, I which unique tags are introduced in each different proximity probe. The combinatorial events generating an in situ PLA signal will harbor a unique identifier tag for each protein interaction. By combining in situ PLA with padlock probes, analysis of signaling activity can be achieved together with genotyping expressed mRNA in fixed tissue sections, retaining the architectural information while providing single-molecule resolution. This “next generation pathology” will enable not only analysis of molecular profiles throughout a tissue section, but might be used to evaluate how cellular communications affects the cellular programs.

 Integrating the Proximity Ligation Assay (PLA) into your Research Program – March 17, 2015

Presented by Joe Roethele, Bethyl Laboratories

The proximity ligation assay (PLA) is a powerful technique for performing in situ analysis of antibody targets in cells or tissues. This antibody-based application is very robust, and will provide unique data on localization and/or interaction of proteins. The webinar will outline the development of PLA, using an approach that is designed to help all researchers—regardless of their level of experience with this application. Topics addressed will include the following: guidelines for getting started, tips on the crucial bioinformatics investigations involved with the planning phase, specific details on the bench-work associated with performing the assay, and troubleshooting suggestions to allow investigators to refine the assay and successfully integrate it into their research program.

 Molecular Annotation of EGFR Signaling-Associated Complexes in Human Cancer – March 26, 2015

Presented by Eric B. Haura M.D., Moffitt Cancer Center

Mass spectrometry and other methods such as yeast two-hybrid can now accurately discern protein complexes or larger protein interactomes in diseases such as cancer, yet it is difficult to forward translate this knowledge into human samples. To overcome this hurdle, we began experiments using proximity ligation assays (PLA) to directly translate protein complexes into human tumor materials. We developed PLA that reflect signaling-associated protein complexes of the epidermal growth factor receptor (EGFR), as a test case, and show the utility of such measurements across a large battery of cell lines, mouse xenograft models, and human lung cancer tissues. Our assay reflects protein complexes between EGFR and GRB2 protein, a key adaptor protein necessary for EGFR pathway activation and coupling to downstream MAPK signaling. We annotated nearly 300 primary xenograft models (PDX) of cancer and show tumor subtype enrichment of EGFR:GRB2 signaling-associated complexes. Furthermore, tumors with abundant levels of EGFR:GRB2 signaling-associated complexes are more likely to respond to anti-EGFR antibody-based therapy. Finally, in 350 lung cancer tissues, across three distinct cohorts of patients, we demonstrate the ability of EGFR:GRB2 protein complexes to segregate tumors and show benefit to EGFR tyrosine kinase inhibitor therapy in patients whose tumors harbor high levels of EGFR:GRB2 signaling-associated complexes. This suggests that annotation of signaling-associated protein complexes in cancer tissues can not only molecularly annotate disease types but may also have predictive capacity for cancer therapeutics. The approach here can be applied to other disease types to better characterize disease based on the presence of particular disease relevant protein complexes. This work opens up the human protein interactome as a new class of molecular markers for disease in a more practical manner. Proteins, encoded by DNA, do not work in isolation but instead function as part of multi-protein complexes that drive both normal and disease physiology. Therefore, annotation of such complexes may allow a new view toward disease characterization. Importantly, while we demonstrate the utility of this approach in cancer, receptor tyrosine kinase signaling and tyrosine kinase inhibitor therapeutics, our approach described here could have utility across a wide spectrum of both signaling-associated complexes and different types of disease, and thus would be attractive to a large audience.

Visualizing DNA Methylation (5mC) on a Specific Genomic Locus (SEPTIN9) in Individual Human Cancer Cells usingIn-situ Hybridization and Proximity Ligation Assays – April 14, 2015

Presented by Dr. Vikas Palhan, Sr. Scientist, Molecular Biotechnology, Sigma-Aldrich

Although there are many techniques to study epigenetic marks such as DNA- and histone-methylation, on a genomic scale, there exists a need in the field to visualize these epigenetic marks at a single genomic locus in individual cells. Such an application requires a highly sensitive detection method. With this aim, a protocol was developed to perform in-situ hybridization followed by proximity ligation assay (a.k.a. Duolink®) and cell imaging to visualize DNA-methylation (5meC) on the SEPTIN9 promoter. SEPTIN9 promoter methylation is a known biomarker for colon cancer. After optimizing cross-linking, cell permeabilization and chromatin accessibility, the genomic specificity was ascertained by hybridizing with a pool of biotinylated-oligo probes that target the CpG islands in the human SEPTIN9 promoter. The Duolink assay was performed using anti-biotin and anti-5meC antibodies, corresponding proximity ligation assay probes, and Far Red detection reagents.

Imaging by fluorescent microscopy revealed two red punctate spots in metastatic prostate cancer DU145 cells (diploid for chromosome 17 – location of SEPTIN9 gene) and three red spots in colon cancer SW480 cells (triploid for Chr17). No signal was observed in normal cells (BJ) or with non-specific oligo probes (LacZ). A decrease in Duolink signal was observed when the DU145 cells were treated with 5-AzaC, a drug known to block DNA-methylation. This proof of concept study will be extended to frozen and formalin fixed paraffin embedded human cancer tissue samples.

Fluorescent imaging data will also be presented from a Duolink assay to monitor the interaction of EZH2 histone methyltransferase with the H3K27me3 epigenetic mark in prostate cancer (DU145) cells. Reduction in the Duolink signal demonstrated inhibition of EZH2 activity by the small molecule inhibitors SAHA and GSK343.

Duolink in Biomedical Science: A Technique for Unraveling the Molecular Mechanisms and Uncovering the Hidden Ultrastructural Features of Gap Junctions – April 28, 2015

Presented by J. Matthew Rhett, PhD, Medical University of South Carolina

Gap junctions (GJs) are large aggregates of intercellular channels that facilitate the diffusion of small molecules and ions between two interacting cells. GJ intercellular channels are formed through the interaction of two half-channels, called hemichannels, composed of oligomerized connexin protein subunits. Both GJs and hemichannels have numerous important physiological and pathological roles in tissue functions including propagation of the action potential in the heart, tumor growth and metastasis, the inflammatory response and adaptive immunity, wound healing, and electrical synaptic transmission in the central nervous system. The most widely expressed connexin isoform is Cx43, and its regulation in the abovementioned processes has been a major focus of GJ research. Over the past two decades protein-protein interaction with the cytoplasmic carboxyl terminus of Cx43 has come to the fore as an endogenous mechanism for controlling the GJ life cycle, channel gating, and channel-independent functions. We have used the Duolink proximity ligation assay (PLA) as a technique to study protein interactions with Cx43 in cultured cells. Two unique aspects of the technology – specifically, subcellular localization and the binary nature of the labeling – in combination with standard immunofluorescent confocal imaging techniques have yielded unexpected insights into GJ ultrastructure, action potential conduction, and the mechanistic regulation of Cx43 trafficking and hemichannel accretion to GJ plaques. In this context, the practical application of, appropriate controls for, and interpretation of Duolink PLAs will be explicated.

Autophagy Machinery Regulates Cell Death Switching: New Insights with Proximity Ligation Assays – June 20, 2017

Presented by Dr. Megan Goodall, University of Colorado, Denver

Although autophagy controls cell death and survival, underlying mechanisms are poorly understood. The importance of this process was highlighted by the award of the 2016 Nobel Prize in Medicine to Yoshinori Ohsumi. However, it is unknown if autophagy simply affects if cells die or also controls other aspects of programmed cell death.

MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. This webcast reports that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TNF-related apoptosis inducing ligand (TRAIL). Surprisingly, this death occurs primarily through necroptosis, not apoptosis. Proximity ligation assays (PLAs) were able to show assembly of the necrosome in association with the autophagy machinery and that it is mediated by p62/SQSTM1 recruitment of RIPK1. Furthermore, the mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo and inhibition at different stages of autophagy is important for cell death.

In this webcast, you will learn:

  • The necrosome associates with autophagy machinery
  • Inhibition of autophagy can promote or inhibit cell death depending on early or late pathway inhibition
  • Autophagy modulates mode of cell death (apoptosis versus necroptosis) through p62/SQSTM1