Name: reash/flash labeling and image analysis of tetracysteine sensor proteins in cells
Uploaded: 2011-08-31T12:00:00
Description: Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

这项工作是由DMH和TDM（NHMRC项目赠款）的赠款。 DMH是Grimwade研究员，由Miegunyah信托基金资助。

1。细胞的制备与ReAsH / FLASH标签<ol><li>使用标准的细胞培养方法，为您感兴趣的细胞株，准备直接在一个活细胞成像幻灯片的转染贴壁细胞培养。 </li><li>转染质粒TC利息根据您所选择的转染法标记的基因。 </li></ol>注意：重​​要的是使用阳性和阴性对照，以评估特异性结合的程度，TC的标签和通道之间的荧光bleedthrough评估时，收集共焦显微。因此，两种颜色（如Flash /樱桃或ReAs...

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Ann. Rev. Biochem. 67, 509-544 (1998).</li><li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-2672 (1998).</li><li>Uttamapinant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorophore+ligase+for+site-specific+protein+labeling+inside+living+cells.">A fluorophore ligase for site-specific protein labeling inside living cells.</a> Proc. Natl. Acad. Sci. USA. 107, 10914-10919 (2010).</li><li>Ignatova, Z., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein+stability+and+aggregation+in+vivo+by+real-time+fluorescent+labeling.">Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.</a> Proc. Natl. Acad. Sci. USA. 101, 523-528 (2004).</li><li>Coleman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+detection+of+prion+protein+with+biarsenical+labeling+and+FlAsH+fluorescence.">Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.</a> Biochem. Biophys. Res. Commun. 380, 564-568 (2009).</li><li>Luedtke, N. W., Dexter, R. J., Fried, D. B., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+polypeptide+and+protein+domain+conformation+and+association+with+FlASH+and+ReAsH.">Surveying polypeptide and protein domain conformation and association with FlASH and ReAsH.</a> Nat. Chem. Biol. 3, 779-784 (2007).</li><li>Ramdzan, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+sensors+that+distinguish+monomeric+proteins+from+oligomers+in+live+cells.">Conformation sensors that distinguish monomeric proteins from oligomers in live cells.</a> Chem. Biol. 17, 371-379 (2010).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., PJ, S. J. R. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Hearps, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biarsenical+dye+Lumio+exhibits+a+reduced+ability+to+specifically+detect+tetracysteine-containing+proteins+within+live+cells.">The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.</a> J. Fluor. 17, 593-597 (2007).</li><li>Adams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biarsenical+ligands+and+tetracysteine+motifs+for+protein+labeling+in+vitro+and+in+vivo:+Synthesis+and+biological+applications.">New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications.</a> J. Am. Chem. Soc. 124, 6063-6076 (2002).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Meth. 2, 905-909 (2005).</li></ol>

一种荧光蛋白和第二个染料的构象属性的方法来标记蛋白定位提供了潜在的映射不同的构象的蛋白质在细胞和改变蛋白质构象的动态的事件累积。 ReAsH / FLASH首次用于哺乳动物细胞视黄酸结合蛋白， 我 4折叠的蛋白质在细胞传感器作为。在这个例子中，绑定到一个TC标签的FLASH设计到细胞视黄酸结合蛋白，我减少了在折叠的形式展开的形式相对荧光量子产率，可以在大肠杆菌中进行?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 8以及μ-幻灯片</td><td> Ibidi </td><td> 80826 </td><td>我们发现这些玻片培养细胞成像特别有用。 </td></tr><tr><td> TC - FLASH II在细胞Tetracysteine​​标签检测试剂盒*绿色荧光*为活细胞成像</td><td> Invitrogen公司</td><td> T34561（FLASH）或T34562（ReAsH） </td><td></td></tr><tr><td>汉克斯“平衡盐溶液</td><td> Invitrogen公司</td><td> 14175-103 </td><td></td></tr><tr><td> 2,3 - 二巯基-1 - 丙醇</td><td> Sigma - Aldrich公司</td><td> D1129 - 5ML </td><td></td></tr><tr><td> 1,2 - Ethanedithiol </td><td> Sigma - Aldrich公司</td><td> 02390 - 25ML </td><td></td></tr></tbody></table>

reash/flash labeling and image analysis of tetracysteine sensor proteins in cells

荧光蛋白染料是蛋白质贩运，本地化和细胞的功能研究的重要工具。虽然荧光蛋白，如绿色荧光蛋白（GFP）已被广泛应用于蛋白质作为融合伙伴跟踪与较小的标签启用新功能的蛋白质，在构象改变，如细 ​​胞研究的利息1蛋白的性质，最近的事态发展和蛋白质协会2，3。一个小标签系统涉及tetracysteine ​​的主题（CCXXCC）基因插入到目标蛋白质，其中结合biarsenical染料，ReAsH（红色荧光）和Flash（绿色荧光），特异性高，即使是在2活细胞。 TC / biarsenical染料系统提供宿主蛋白的荧光蛋白比，这使一些新的方法来衡量的构象变化和蛋白质的相互作用4-7远不如立体的约束。我们最近开发的传感器齐聚细胞表达突变亨廷顿在一个新颖的TC标签应用程序发生突变时，聚集在神经元在亨廷顿病7。亨廷顿是两个荧光染料，荧光蛋白跟踪蛋白的位置，第二一个TC标签，它仅在单体结合biarsenical染料标记。因此，蛋白质和biarsenical染料反应之间的共定位的变化，使细胞内的空间映射微观低聚物含量。在这里，我们描述如何标注训练班标签蛋白融合在生活的哺乳动物细胞的FLASH或ReAsH和如何量化两色荧光（樱桃/ FLASH，CFP / Flash或GFP /荧光蛋白（樱桃，GFP或CFP） ReAsH组合）。

Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

biarsenical染料Flash和ReAsH特异性结合蛋白质tetracysteine​​图案，并在活细胞中的蛋白质可以选择性地标签。最近这个标签的战略已被用于开发传感器用于不同的蛋白质的构象或低聚国家。我们描述了标签的方式和方法，定量分析具有约束力。

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

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科研

生物学

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Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

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ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

ReAsH / FLASH标签和图像分析Tetracysteine传感器蛋白在细胞中