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Detection and Visual Localization of Individual Cellular Proteins Using a Proximity Ligation Assay (Presentation)Download
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February 03, 2016
Authors: Paul Held and Xavier Amouretti, BioTek Instruments, Inc. Winooski, VT USA
With the mapping of the human proteome the understanding of protein interaction and localization in mammalian cells grows ever more complex. The complexity of the proteome and the understanding of its inner workings create a need for more precise and accurate ways of detecting targets. Besides general detection and localization of a protein, it is necessary to investigate its interaction partners, posttranslational modifications or its role in protein complexes in order to understand the complete role a particular protein plays.
Proximity ligation assays (in situ PLA) combine the specificity of antibody based detection of antigenic epitopes with a signal amplification that allows the visualization of components through fluorescence microscopy [1-2]. Proximity ligation assay is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions and modifications with high specificity and sensitivity. Protein targets can be detected and localized with single molecule resolution and quantified in unmodified cells and tissues.
BioTek’s Cytation™ 5 has a number of features that enable Duolink® imaging. Four separate LED positions allow for multiplex fluorescence imaging using a number of different magnification microscope objectives. Besides identification of the protein(s) of interest, counterstaining for cytoplasmic markers or nuclei provide cellular location information. In addition the imager holds 6 objectives with magnification up to 60X. Gen5™ software provides autofocusing of cells in microplates, capturing of images with both automatic or user defined parameters (LED intensity, CCD gain, integration time, etc.) and cellular analysis algorithms that allow for cell segmentation and cell counting. The Gen5 software used to control reader function is also capable of significant automated image analysis. Image analysis can be used to count numbers of cells that meet threshold and size criteria.
Cytation™ 5 Cell Imaging Multi-Mode Reader