( META...)High-content screening of GPCR activation with MetaXpress, AcuityXpress and the Transfluor assay system
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High-content screening of GPCR activation with MetaXpress, AcuityXpress and the Transfluor assay system
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METAXPRESS APPLICATION NOTE
By Susan Catalano, Ph.D., Acumen Pharmaceuticals, Sylvia de Bruin, Joe Bosworth, Ph.D.,
Neal Gliksman, Ph.D., Kimberly Best, Ph.D. and Paula Rickert, Ph.D., Molecular Devices Corporation,
1311 Orleans Drive, Sunnyvale, CA 94089. The Transfluor Assay, validated on over 90 different GPCRs, uses a common pathway independent of downstream G-protein signaling which is shared by virtually all GPCRs, representing all classes of GPCRs and G proteins. The assay uses image analysis to quantitate internalization of GFP-tagged beta-arrestin that associates with the receptor of interest following activation.1 (See Figure 1.)
Unstimulated cells display diffuse cytoplasmic beta-arrestin-GFP fluorescence.
When an over-expressed GPCR is activated, beta-arrestin targets the receptor
for internalization, resulting in the appearance of small fluorescent
clathrin-coated pits. If the receptor has a high affinity for beta-arresti Related biology technology : 1. siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005) 2. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors 3. Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening 4. Inactivation of Contaminating Templates in PCR Using High Intensity Ultraviolet Light 5. Enzyme Immunoassay for Studying Intracellular Levels of cAMP 6. LiquiChip Kits for bead-based cytokine and kinase assays 7. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay 8. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A 9. 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