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Duolink® PLA Fluorescence Protocol

Merck
16 Oct 2017

This protocol describes the use of Duolink® PLA reagents for the immunofluorescent detection, visualization, and quantification of individual proteins, protein modifications, and protein interactions in tissue and cell samples. Learn more about Duolink® PLA technology.

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Duolink In Situ Red Starter Kit Mouse/Rabbit

Merck

Application Duolink® proximity ligation assay(PLA®) allows for endogenous detection of protein interactions, post translational modifications, and protein expression levels at the single molecule level in fixed cells and tissue samples. Follow the Duolink® In Situ Fluorescence Protocol to use this product. A set of short instructionsis also available. Visit our Duolink® PLA Resource Center for information on how to run a Duolink®experiment, applications, troubleshooting, and more. To perform a complete Duolink® PLA in situ experiment you will need two primary antibodies (PLA, IHC, ICC or IF validated) that recognize two target epitopes.This starter kit supplies all other necessary reagents for 30 Duolink® PLA reactions, which include a pair of PLA probes (Anti-Rabbit PLUS and Anti-Mouse MINUS), red detection reagents, wash buffers, and mounting medium. Note that the primary antibodies must come from the same species as the Duolink® PLA probes. Analysis is carried out using standard immunofluorescence assay equipment. Specificity The Duolink® In Situ Red Starter Kit Mouse/Rabbit requires one primary antibody from mouse and one primary antibody from rabbit. Red fluorescence detection reagents are often used with Texas Red® filter. Application Note  Two primary antibodies raised in different species are needed. Test your primary antibodies (IgG-class, mono- or polyclonal) in a standard immunofluorescence (IF), immunohistochemistry (IHC) or immunocytochemistry (ICC) assay to determine the optimal fixation, blocking, and titer conditions. Duolink® PLA in situ reagents are suitable for use on fixed cells, cytospin cells, cells grown on slide, formalin-fixed, paraffin embedded (FFPE), or tissue (fresh or frozen). No minimum number of cells is required. Features and Benefits No overexpression or genetic manipulation required High specificity (fewer false positives) Single molecule sensitivity due to rolling circle amplification Relative quantification possible No special equipment needed Quicker and simpler than FRET Increased accuracy compared to co-IP Publication-ready results

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AntibodiesAntibodies are used in techniques such as confocal and fluorescence microscopy, flow cytometry, ELISA, ELISPOT, immunohistochemistry, western blotting and immunopreciptation. Select specific antigen reactivity, high specific affinity, low non-specific binding, monoclonal or polyclonal, primary or secondary antibodies and associated conjugates such as an enzyme or dye for visualization.Cell / Tissue CultureCell culture or tissue culture is used to study the biology of cells or tissues and to isolate cellular products in an environment which can be manipulated and well defined. Accurately control your culture environment with bioreactors or culture incubators, bind your cells to a surface or together with an extracellular matrix. Distinguish cell types with differential media or proliferate cells with certain characteristics using selective media. Enrich your media with supplements such as growth factors, sera and vitamins. Find the best cell and tissue culture products, kits and equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.OligonucleotidesOligonucleotides are small nucleic acid polymers, usually less than 20 bases in length. Oligonucleotides can be made via enzymatic cleavage or more commonly by chemical synthesis with polymerases. Their use includes FISH, southern blots, microarrays and as primers in PCR. High fidelity synthesis kits and detection systems are available for easy production and detection, respectively.ProteomicsProteomics is the systemic bioinformatics study of proteins and amino acids, including their structure, size, function and identification. Tools used in proteomics include chromatography, blotting and gels, protein arrays, mass spectrometry and ELISA and associated analysis software. Analyzers and proteomic systems should be sensitive, high resolution, fast and may be automated for high-throughput.Cell-Based AssaysCell-based assays are used to monitor the presence, quantity and activities of a desired cellular analyte including drug molecules or biomarkers. This can reveal information on cell health (apoptosis, cytotoxicity, viability and proliferation assays), cell metabolism, cell migration and cell signaling mechanisms. Find the best cell-based assay products, kits and equipment with our peer reviewed product directory: compare products, check customer reviews and receiving pricing direct from manufacturers.BiomarkersBiomarkers are biological markers which can be measured and evaluated to indicate a biological state. The use of biomarkers in research and diagnosis can indicate a normal or disease state or drug response of cells / tissues. Biomarkers include genetic markers, cell surface markers such as antigens, antibodies or receptors and secreted molecules such as cytokines. An assay system is required for identification of biomarkers. :ImmunoassayImmunoassays are laboratory tests that utilize antibody-antigen interactions to detect specific substances. They are widely used in diagnostics for detecting diseases, drug testing, and environmental monitoring. These assays offer rapid, sensitive, and precise detection of biomarkers, making them invaluable in clinical and research labs. Browse our peer-reviewed product directory to compare immunoassay systems, read user reviews, and get pricing directly from manufacturers.ImmunofluorescenceProtein InteractionsProtein interactions are essential for understanding cellular processes, as proteins work together to carry out biological functions. Studying protein interactions can provide insights into disease mechanisms, drug development, and cell signaling pathways. Explore the best tools for protein interaction research in our peer-reviewed product directory; compare products, check reviews, and get pricing directly from manufacturers.Post-Translational ModificationsIn Situ HybridizationProtein BiologyThe analysis of protein expression, identity and function is vital for many areas of life science research and drug discovery. Some of the most commonly used techniques in protein analysis include Western blotting, electrophoresis and mass spectrometry.

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