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Cell-Based Applications of Living Colors® Proteins Valuable tools in multicolor screening applications • Easy detection compatible with diverse instrument platforms including the BD™ Pathway HT confocal bioimaging system A Untreated B Treated (bottom of cell) C Treated (middle of cell) • Ideally suited for subcellular protein trafficking and highcontent screening assays • No need for added cofactors or substrates Cell-based assays have undergone tremendous development in recent years as current screening technologies evolve toward cost-effectiveness and biological significance. This shift in focus strongly emphasizes more relevant, informative, and complex cell-based systems over the traditional in vitro-based assays. Fluorescent proteins, historically wellestablished tools for noninvasive and real-time studies in live cells, are now being used in a variety of reagent-free homogenous assays that address current screening needs. The spectral properties of fluorescent proteins make them extremely well suited for multicolor applications in fluorometry, flow cytometry, and fluorescence microscopy—fluorescent proteins have great potential in both optimizing and accelerating drug discovery and screening processes. We offer a panel of Clontech Novel Fluorescent Proteins (NFPs) suited for precisely these applications. They consist of the Reef Coral Fluorescent Proteins (RCFPs)—AmCyan1, ZsGreen1, ZsYellow1, DsRed, AsRed2, and HcRed1, as well as our new monomeric DsRed-Monomer (red) and AcGFP1 (green) proteins (1–3). DsRed-Monomer is the only commercially available red monomeric fluorescent protein suitable for dual color fusion applications with AcGFP1. Figure 1. GPCR activation monitored by Living Colors DsRed2 fusion proteins in the Transfluor assay. U-2 OS cells were cotransfected with the ß2-adrenergic receptor and a ß-arrestin-2-DsRed2 fusion protein and treated for 30 min with 1 µM isoproterenol. Cells were analyzed by confocal microscopy at 63X magnification. Panel A. Untreated cells. Panels B and C. Images of stimulated cells were taken at either the interface of the cell and the plate, or at a cross section through the cell. Our comprehensive collection of distinct, novel fluorescent proteins covers an extensive emission spectrum that ranges from 489 nm (AmCyan1) to 618 nm (HcRed1) (4, 5). No cofactors or substrates need to be added for detection, which further simplifies and streamlines your experiments. Wide range of applications Our fluorescent proteins are powerful tools for establishing a wide range of assays (reviewed in 4). RCFPs and AcGFP1 have been used successfully as fusion partners when fused to a targeting signal, and are ideal markers for cells and organelles (6–7). Monomeric AcGFP1 and DsRed-Monomer are the best candidates for studies requiring full-length protein fusions—they perform extremely well in fusions with full-length actin, tubulin, and insulin (data not shown). Furthermore, RCFPs have been successfully expressed in a diverse range—plant to animal—of whole body organisms (1; data not shown). RCFPs that are bright and/or available in a destabilized format, such as ZsGreen1, HcRed1, and DsRedExpress, are also perfect transcriptional reporters (4, 6). The RCFPs, in particular ZsGreen, have also been shown to be powerful and sensitive biosensors for monitoring live-cell proteasome activity as well as markers for in vitro migration assays modeling metastatic tumor invasion (8; data not shown). Clontech Laboratories, Inc. • www.clontech.com CR682048 Ideal for high-content screening assays The examples highlighted here complement the previously published uses of Clontech NFPs as specialized screening tools. NFPs can be used to establish reliable, high-content cell-based assays such as monitoring G-protein coupled receptor (GPCR) activation. Use of a DsRed2 fusion to the ß-arrestin-2 regulatory protein provides an elegant Norak Transfluor™ assay for monitoring ß2-adrenergic receptor activation (Figure 1). In the absence of agonist, the DsRed2-ß-arrestin-2 fusion protein is distinctly localized to the cytoplasm. Upon agonist stimulation, the fusion moves to the plasma membrane where it participates in receptor desensitization and clathrin-coated pit internalization. The formation of coated pits indicates activation of the ß2-adrenergic receptor. Reprinted from Clontechniques January 2005 Cell-Based Applications…continued A B Figure 2. Four color visualization of Living Colors Novel Fluorescent Proteins using the BD™ Pathway Confocal Bioimager. Panel A. HEK 293 clonal cell lines stably expressing AmCyan1, ZsGreen1, ZsYellow1, or HcRed1 were mixed and plated into the same well and then imaged at 20X magnification using the BD™ Pathway Bioimager. Chroma Technology filter sets were used to separate the signal of each individual fluorescent protein (HQ440/30, 470DCXR, and HQ488/35 for AmCyan1; HQ488/10, 84100 beam splitter, and HQ540/50 for ZsGreen1; HQ500/40, 530DCLP, and HQ555/40 for ZsYellow1; and HQ575/50x, Q610LP, and HQ640/50m for HcRed1). Panel B. HEK 293 cells were transiently transfected with constructs encoding AmCyan1-Peroxi (peroxisome), ZsYellow1-Nuc (nucleus), and HcRed1-Mito (mitochondria) fusion proteins. Slides were imaged at 60X magnification with an oil objective using the method described above. Multiple NFPs can identify distinct cell subpopulations and intracellular structures Notice to Purchaser For all licensing information, visit www.clontech.com Multicolor detection within a mixed population of cells expressing AmCyan1, ZsGreen1, ZsYellow1 or HcRed1 can be performed using a confocal microscopy platform such as the BD™ Pathway HT Confocal Bioimager (Figure 2, Panel A). Our NFPs can also be used to label and easily distinguish subcellular organelles and compartments from one another in live cells (Figure 2, Panel B). Clontech Fluorescent Proteins, with their multiplexing capabilities and uniquely suited qualities, should play an ever increasing role in the development of even more sophisticated cell-based drugdiscovery and screening processes. Living Colors Products Size Cat. No. pAcGFP1-N1 Vector 20 µg 632469 pAcGFP1-C1 Vector 20 µg 632470 pAmCyan1-N1 Vector 20 µg 632442 pAmCyan1-C1 Vector 20 µg 632441 pAsRed2-N1 Vector 20 µg 632449 pAsRed2-C1 Vector 20 µg 632450 pDsRed2-N1 Vector 20 µg 632406 pDsRed2-C1 Vector 20 µg 632407 pDsRed-Express-N1 Vector 20 µg 632429 pDsRed-Express-C1 Vector 20 µg 632430 pDsRed-Monomer-N1 Vector 20 µg 632465 pDsRed-Monomer-C1 Vector 20 µg 632466 pHcRed1-N1/1 Vector 20 µg 632424 pHcRed1-C1 Vector 20 µg 632415 pZsGreen1-N1 Vector 20 µg 632448 pZsGreen1-C1 Vector 20 µg 632447 pZsYellow1-N1 Vector 20 µg 632445 pZsYellow1-C1 Vector 20 µg 632444 References 1. Matz, M. V., et al. (1999) Nature Biotechnol. 17:969–973. Erratum in: Nature Biotechnol. (1999) 17:1227. 2. Gurskaya, N. G., et al. (2001) FEBS Lett. 507:16–20. 3. Gurskaya, N. G., et al. (2003) Biochem. J. 373:403–408. 4. Haugwitz M., et al. (2003) Genet. Eng. News 23(21). 5. Verkhusha, V. V. & Lukyanov, K. A. (2004) Nature Biotechnol. 22(3):289–296. 6. Reef Coral Fluorescent Protein Vectors (July 2003) Clontechniques XVIII(3):67. 7. BD Living Colors™ HcRed1 Localization Vectors (July 2003) Clontechniques XVIII(3):8. 8. BD Living Colors™ Cell Lines (April 2004) Clontechniques XIX(2):2–4. Clontech Laboratories, Inc. • www.clontech.com Living Colors® Related Products • Anti-RCFP Polyclonal Pan Antibody (Cat. No. 632475) • Full-Length ZsGreen Polyclonal Antibody (Cat. No. 632474) • DsRed Monoclonal Antibody (Cat. Nos. 632392 & 632393) • DsRed Polyclonal Antibody (Cat. No. 632397) • HcRed Polyclonal Antibody (Cat. No. 632452) • BD™ Pathway Bioimager (inquire) For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech, Clontech logo, and all other trademarks are the property of Clontech Laboratories, Inc. Clontech is a Takara Bio Company. Copyright 2006. Reprinted from Clontechniques January 2005