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Duolink® PLA Troubleshooting Guide

Merck
6 Nov 2017

Read this application note for tips, tricks and support for using Duolink® proximity litigation assay (PLA) kits.

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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.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.Fluorescence MicroscopyFluorescence microscopy has become an essential tool in biology, as well as in materials science. The application of many fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity. Using multiple fluorescence labels, different probes can simultaneously identify several target molecules.Cell Based AssaysCell-based assays use living cells to measure biological responses to various treatments or stimuli. They are essential for drug discovery, toxicology testing, and understanding cellular mechanisms. These assays are used to assess cell viability, proliferation, gene expression, and other cellular activities. Browse our peer-reviewed product directory to find the best cell-based assay tools and reagents, compare products, read customer reviews, and get pricing directly from manufacturers.Bright Field MicroscopyProtein 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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Duolink® PLA Troubleshooting Guide