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High-Throughput Screening of Protein Localization

Lee Opresko and Marianne Sowa, Principal Investigators


Researchers at PNNL have designed a high-speed, high-sensitivity confocal microscope to capture simultaneously output from two intensified CCD cameras.

The ability to rapidly localize proteins expressed in cells would greatly enhance our knowledge of potential interaction complexes as well as provide information about the behavior of proteins in response to external stimuli in their natural environment (the intact cell). To this end, Pacific Northwest National Laboratory's (PNNL's) High-Throughput Screening of Protein Localization research team is implementing a high-throughput visualization system that couples a high-speed confocal microscope capable of collecting two-color images in real time with reference mammalian cells that contain fluorescently labeled subcellular compartments. This visualization system is being combined with high-speed data acquisition and analysis and high-throughput cloning and retroviral propagation to express fluorescently labeled gene products in the reference cells.

Fluorescent micrographs, graphic
Fluorescent micrographs of two different human mammary epithelial cell (HMEC) lines that express either endosomal- or membrane-targeted monomeric red fluorescent protein (mRFP). Cell line A1L5 is a subclone of line A1. These images show there is more membrane- and endosome-specific localization in A1L5 cells. Neither marker is optimal in either cell type. Click for a larger version.

As the result of this project, PNNL staff are developing a high-throughput screening capability. This system will be tested for its ability to collect and analyze a large data array from visual input and to do so in an automated fashion. Protocols will be optimized and standardized for the entire process of high-throughput screening, including high-throughput DNA cloning, retroviral production, gene expression, and analysis.

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