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Cyto-Cell Membrane Translocation BioApplication |
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Catalog No. S50-0012-2
DescriptionCell signaling proteins translocate to different cell compartments in response to specific inducers, to modulate and regulate various cellular functions. Some of these targets redistribute between the cytoplasm and the cell membrane in response to agonist binding. The Cytoplasm to Cell Membrane Translocation BioApplication measures the translocation of proteins from the cytoplasm to the cell membrane and vice versa. This BioApplication provides quantitative information on protein translocation using images of fixed cells stained with fluorescent markers. The BioApplication reports several cell-level and well-level output features that enable users to make decisions in their High Content Screening campaigns, including: quantity of the target protein at the cell membrane in comparison to the amount in the cytoplasm, co-localization of the target protein at the cell membrane, and percentage of cells in which the translocation event exceeded user-defined thresholds. The Cyto-Cell Membrane Translocation BioApplication is therefore useful for screening compounds that are potential agonists or inhibitors of protein translocation from the cytoplasm to the cell membrane or vice versa. Thermo Fisher Scientific has successfully demonstrated the measurement of protein kinase C alpha (PKCa) translocation from the cytoplasm to the cell membrane using this BioApplication in conjunction with the PKCa Activation HitKitTM HCS Reagent Kit. The PKC family is a group of related signal transduction proteins, and their activation is one of the earliest events leading to a variety of cellular responses including cytokine secretion, differentiation, proliferation, muscle contraction, and modulation of membrane excitability. PKCa is believed to play an important role in the development and growth of cancer cells. Inhibition of PKCa production is the basis for therapeutic candidates currently in clinical trials for the treatment of non-small cell lung cancer. PKCa may also underlie the cellular mechanism leading to inflammatory lung vascular injury and adult respiratory distress syndrome. HeLa cells grown on standard 96-well microplates were stimulated, fixed, and fluorescently labeled to identify PKCa (Figure 2). 
PKCa is diffusely and evenly stained throughout the cytoplasm in the absence of activation. In response to activation with a known agonist, phorbol 12-myristate 13-acetate (PMA), PKCatranslocates to the cell membrane. Figure 3 shows dose response curves for PKCa activation in response to PMA treatment of HeLa cells. The three curves represent three distinct assay output features that can be used to measure translocation to/from the cell membrane. The extent of translocation can be quantified in several ways with the Cyto-Cell Membrane Translocation BioApplication, making it a flexible BioApplication useful for different targets. In addition, a second target can be measured at the same time thus enabling a true High Content Screen for two biomolecules of interest. This is especially useful for specificity studies. Therefore, the Cyto-Cell Membrane Bioapplication from Cellomics, Inc. is a user-friendly solution for functional screening for modulators of cytoplasm to cell membrane translocation.
Features- BioApplication automatically calculates and reports the distribution of the target of interest
- BioApplication measures and reports translocation features for two targets, allowing multiplexing of assays
- Assay is performed on intact cells and requires no cell lysis, protein purification, generation stable transfected cell lines, or radioactivity
- Validated image analysis algorithm shortens assay development time
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