The authors thank Drs. Steven Chu and Joe W. Gray for helpful discussions, Henry Marr for his initial work on the BiFC-PALM project, and Alexis Shoemaker for her technical assistance. This work is supported by startup funds to X.N. from OHSU. Research in the Nan laboratory was also supported by NIH 5U54CA143836-05, the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI company.

<ol>
	<li>Braun, P., Gingras, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+protein-protein+interactions:+From+egg-white+to+complex+networks.">History of protein-protein interactions: From egg-white to complex networks.</a> Proteomics. 12, 1478-1498 (2012).</li><li>Lasserre, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raft+nanodomains+contribute+to+Akt/PKB+plasma+membrane+recruitment+and+activation.">Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation.</a> Nat. Chem. Biol. 4 (9), 538-547 (2008).</li><li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol. Cell. 9 (4), 789-798 (2002).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+analysis+as+a+probe+of+protein+interactions+in+living+cells.">Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.</a> Annu. Rev. Biophys. 37, 465-487 (2008).</li><li>Kodama, Y., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC):+a+5-year+update+and+future+perspectives.">Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives.</a> Biotechniques. 53, 285-298 (2012).</li><li>Lingwood, D., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Rafts+As+a+Membrane-Organizing+Principle.">Lipid Rafts As a Membrane-Organizing Principle.</a> Science. 327 (5961), 46-50 (2009).</li><li>Tian, T., Harding, A., Inder, K., Plowman, S., Parton, R. G., Hancock, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+nanoswitches+generate+high-fidelity+Ras+signal+transduction.">Plasma membrane nanoswitches generate high-fidelity Ras signal transduction.</a> Nat. Cell Biol. 9 (8), 905-914 (2007).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91 (11), 4258-4272 (2006).</li><li>Nickerson, A., Huang, T., Lin, L. J., Nan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivated+Localization+Microscopy+with+Bimolecular+Fluorescence+Complementation+(BiFC-PALM)+for+Nanoscale+Imaging+of+Protein-Protein+Interactions+in+Cells.">Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) for Nanoscale Imaging of Protein-Protein Interactions in Cells.</a> PloS one. 9 (6), e100589 (2014).</li><li>Manley, S., Gillette, J. M., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+photoactivated+localization+microscopy+for+mapping+single-molecule+dynamics.">Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics.</a> Methods Enzymol. 475, 109-120 (2010).</li><li>Shyu, Y. J., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+complementation:+an+emerging+tool+for+biological+research.">Fluorescence complementation: an emerging tool for biological research.</a> Trends Biotechnol. 26 (11), 622-630 (2008).</li><li>Fan, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+mCherry+as+a+new+red+bimolecular+fluorescence+complementation+system+for+visualizing+protein-protein+interactions+in+living+cells.">Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.</a> Biochem. Biophys. Res. Commun. 367 (1), 47-53 (2008).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Gomez-Martinez, M., Schmitz, D., Hergovich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+human+cell+lines+with+Tetracycline-inducible+(Tet-on)+shRNA+or+cDNA+expression.">Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.</a> J. Vis. Exp. March. (73), e50171 (2013).</li><li>Robida, A. M., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+analysis+of+inducible+protein+interactions:+effects+of+factors+affecting+protein+folding+on+fluorescent+protein+fragment+association.">Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association.</a> J. Mol. Biol. 394 (3), 391-409 (2009).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+biosensors+based+on+dimerization-dependent+fluorescent+protein+exchange.">Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange.</a> Nat. Methods. 12 (3), (2015).</li><li>Seidman, C. E., Struhl, K., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+plasmid+DNA+into+cells.">Introduction of plasmid DNA into cells.</a> Curr. Protoc. Protein Sci. Appendix 4, 4D (2001).</li><li>Morrison, S. L., Coligan, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+frozen+bacterial+stocks.">Preparing frozen bacterial stocks.</a> Curr. Protoc. Immunol. Appendix 3, Appendix 3M (2001).</li><li>Campeau, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+viral+system+for+expression+and+depletion+of+proteins+in+mammalian+cells.">A versatile viral system for expression and depletion of proteins in mammalian cells.</a> PloS One. 4 (8), e6529 (2009).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnol. 8 (91), (2008).</li><li>Deschout, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+and+accurately+localizing+single+emitters+in+fluorescence+microscopy.">Precisely and accurately localizing single emitters in fluorescence microscopy.</a> Nat. Methods. 11 (3), 253-266 (2014).</li><li>Nan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+superresolution+imaging+allows+quantitative+analysis+of+RAF+multimer+formation+and+signaling.">Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.</a> Proc. Natl. Acad. Sci. USA. 110 (46), 18519-18524 (2013).</li><li>Chenouard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+comparison+of+particle+tracking+methods.">Objective comparison of particle tracking methods.</a> Nat. Methods. 11 (3), 281-289 (2014).</li><li>Persson, F., Lind&#233;n, M., Unoson, C., Elf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracting+intracellular+diffusive+states+and+transition+rates+from+single-molecule+tracking+data.">Extracting intracellular diffusive states and transition rates from single-molecule tracking data.</a> Nat. Methods. 10 (3), 265-269 (2013).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+and+tracking+of+protein&#8211;protein+interactions+in+sub-diffraction+cellular+space.">Super-resolution imaging and tracking of protein&#8211;protein interactions in sub-diffraction cellular space.</a> Nat. Commun. 5, 1-8 (2014).</li><li>Xia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+reveals+structural+features+of+EB1+in+microtubule+plus-end+tracking.">Super-resolution imaging reveals structural features of EB1 in microtubule plus-end tracking.</a> Mol. Biol. Cell. 25 (25), 4166-4173 (2014).</li></ol>

The authors have nothing to disclose.

附设一直是常用的技术，用于检测和细胞可视化质子泵抑制剂，而PALM是最近单分子超分辨率显微镜技术，使完整的生物样品的纳米级成像。附设与Palm的结合纳米的空间分辨率和单分子敏感性细胞内获得的生产者价格指数的选择性成像。附设掌扩展这两种技术的效用，而这表现在这项工作中，显示了在揭示其天然细胞环境的生产者价格指数的分子细节巨大潜力。尤其是，纳米级分辨率允许特定质?...

蛋白质-蛋白质相互作用（质子泵抑制剂）是基本的生物学1和经由跨许多时间和长度尺度时空机制紧密调节。研究细胞在细胞膜上的信号，例如，已揭示动态，纳米级的空间隔室，以促进特定质子泵抑制剂和细胞过程2。因此，探讨生物系统的生产者价格指数有足够的空间和时间分辨率，高特异性和高灵敏度的能力，关键是要实现生物的机械理解。 双分子荧光互补（附设）是用于可视化的生产者价格指数在细胞与亚细胞分辨率和活细胞相容性3存在的少数手段之一- 5。该技术是相对简单的，并且涉及一个荧光蛋白为两个非荧光片段的分裂;当遗传标记，以两个相互作用的蛋白质和使得接近，所述片段可重组，以形成一个完整的荧光蛋白，产生荧光信号。如果设计得当，附设探头不应该自发重组在没有生产者价格指数。这样，在一个附设测定荧光信号将仅出现在质子泵抑制剂的存在下，使质子泵抑制剂以高特异性的直接可视化。使用荧光作为读出的附加好处是高灵敏度，亚细胞的分辨率，并具有高通量和高含量筛选测定法，等等的兼容性。对于这些好处，已经开发了许多基于不同母体的荧光蛋白质附设探针。正如在基于常规的光学显微镜所有其它检测技术，但是，附设的空间分辨率由光的衍射限于〜250纳米。这使得它的挑战，研究生产者价格指数的调控在纳米尺度，对此，由脂质筏6先前提到并举例第二拉斯纳米团簇7，是一个重要的尺度来理解许多细胞过程，如信号。 附设已结合光敏定位显微镜（PALM）8,9克服这个限制的空间分辨率的成像生产者价格指数10。 PALM是绕过通过随机激活和单荧光分子subdiffractive本地化荧光成像衍射极限的近超分辨率显微镜技术。在每个激活周期，荧光分子发出几百到几千光子并产生了一个单分子图像在检测器上。当图像衍射极限（〜在宽度250纳米），其质心可以以更高的精确度来确定，通常在10-50纳米根据所检测的光子数的顺序。通过激活和定位于样品中的每个荧光分子，高分辨率图像可以被重建。 PerformeD于活细胞，单分子跟踪（SMT-）PALM进一步允许采集的数千蛋白扩散轨迹从单电池 11。重要的是，PALM使用专门的荧光探针，如光活化荧光蛋白（PA-FPS）来实现随机启动。由于两个附设和Palm使用荧光蛋白，他们通过分割PAmCherry1，常用的PA-FP对于Palm，变成氨基酸159和160之间的两个片段合并。 基于分裂PAmCherry1的附设系统显示来自两个片段自发重建低背景信号。当遗传标记，以一对相互作用的蛋白质，这两个片段（RN =残基1-159; RC =蛋氨酸+残基160-236）再生有效，即使在37℃和无孵育在较低温度下，以形成完整的PAmCherry1蛋白，其是不为其他附设对12如父mCherry 13的情况。 Furthermo重，再溶解后PAmCherry1蛋白保留了母体PAmCherry1的光物理性质，诸如高对比度，中等光子产率，和快速光活化，等等，这对于精确的单分子的定位和高分辨率的PALM成像是至关重要的。 在这个协议中，利用附设掌的成像U2OS细胞的Ras-Raf的交互，采用分体式PAmCherry1（ 图1A）中描述。第一步是设计构建用于表达PAmCherry1片段（ 即 ，RN和RC）和感兴趣的蛋白质之间的融合蛋白。从理论上讲，对于每对候选蛋白（A和B）的，有八个对融合蛋白进行测试：RN-A / RC-B; RN-A / B-RC; RC-A / RN-B; RC-A / B-RN; A-RN / RC-B; A-RN / B-RC; A-RC / RN-B;和A-RC / B-RN。这个过程通常可以通过考虑候选蛋白的结构或生物化学特性被简化。在的Ras的情况下，该蛋白质是翻译后修饰的C-末端CAAX盒（C =半胱氨酸; A =脂族; X =任何），在这之后AAX基序被裂解掉。因此，RN或RC只能融合到的Ras的N-末端;这降低了融合蛋白对四个（图1B）的数目。对于Raf的，所述的Ras结合结构域（RBD残基51-131）的使用，并且能够进行标记两端。生成这四个融合配置：RN-KRAS / RC-RAF RBD; RN-KRAS / RAF RBD-RC; RC-KRAS / RN-RAF RBD;和RC-KRAS / RAF RBD-RN。 此外，片段和所关心的蛋白之间的连接体将需要被考虑。约10个氨基酸柔性接头常常被用来作为它提供了足够的自由互补发生。一个这样的接头是（GGGGS）×2，虽然有许多人已成功地应用，包括从一个多克隆位点（MCS）14生成的随机序列。所述接头的长度可能需要被优化，取决于不感兴趣的蛋白质和当相互作用及其取向的大小克。 所述PAmCherry1片段被包含在一小克隆骨干具有侧翼的MCS（参见材料清单）。目的基因可以通过限制性位点或具有结扎无关的方法插入。之后克隆和序列验证，表达盒转移到使用重组酶反应中，克隆过程具有高保真和高效率的表达载体。 接着，将得到的表达构建体转染到靶细胞系，或者，如果一个稳定的细胞系是理想的，包装成慢病毒为感染靶细胞系。瞬时转染允许附设配置快速验证，但是潜在的问题必须注意。使用的化学品经常染强调细胞，导致高自发荧光;而采用全内反射荧光（TIRF）microsco的吡啶可以通过限制所述照明体积，TIRF理想当质子泵抑制剂发生在细胞膜上缓解这个背景信号。此外，瞬时转染往往导致高水平的蛋白表达的，远超过那些内源蛋白质，这可能会导致在检测所述质子泵抑制剂工件。因此，建议的稳定细胞株后的适当附设配置已通过初步测试被确定来建立。稳定细胞株也有通过多西环素诱导15为可调表达的潜力。 感染后，将细胞进行选择用的抗生素，通常嘌呤霉素和新霉素，每个构建体。对于样品制备，图像采集和数据分析的后续步骤将在详细的协议进行说明。 采用这种方法，纳米簇中的的Ras-Raf的RBD附设-PALM图像形成例行观察（图2A &lt;/ STRONG&gt;）。一致，对的Ras-Raf的RBD SMT-PALM轨迹显示在扩散状态（图2B-D）的一个非均匀分布。这些结果表明，在多个状态存在于细胞膜上的Ras-Raf的络合物，据推测为单体和簇，具有潜在的生物学意义。这项工作表明在质子泵抑制剂在细胞与纳米空间分辨率和单分子灵敏度，这将是很难获得与常规附设，荧光共定位，或荧光共振能量转移（FRET）的选择性成像附设-PALM的功率。 当设计附设实验和解释的结果，要记住的附设过程是不可逆在大多数情况下，包括分裂PAmCherry1是重要的。一旦两个片段结合并形成一个完整的PAmCherry1蛋白，这两个片段之间的联系，因此，PPI成为永久性的。这限制了使用附设和附设-PAL的M表示监视质子泵抑制剂的动力学（即，结合动力学，而不是蛋白质复合物一旦被形成在扩散动力学），有时甚至有可能导致到PPI络合物的错误定位。 一个附加的需要考虑的因素在设计附设-PALM实验是延迟在发色团成熟所固有的荧光蛋白质。一旦两种蛋白质相互作用和在FP片段聚集在一起，它们通常重折叠的秒（60秒的一半时间为EYFP 3）的数量级。然而，随后的发色团成熟和荧光信号开发分钟的量级。虽然荧光可在拆分 ​​金星，快速折叠YFP变种重建后10分钟内检测，半时间成熟，一般往往是约60分钟16。分裂PAmCherry1观察到有类似的速率。因此，附设及附设掌是监视实时PPI动力学目前糟糕的选择;其他的我thods，如FRET和最近开发的二聚化相关的荧光蛋白17，可能更适合用于此目的。

<table>
	<tbody>
		<tr>
			<td>TIRF Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60X oil immersion TIRF objective with 1.49 NA</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>&#160;Di01-R405/488/561/635-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm filter</td>
			<td>Semrock</td>
			<td>FF01-525/45-25</td>
			<td></td>
		</tr>
		<tr>
			<td>405/561 nm notch filter</td>
			<td>Semrock</td>
			<td>NF01-405/488/568-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature and CO2 controlled stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry1_1-159-MCS</td>
			<td>Addgene</td>
			<td>60545</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry160-236-MCS</td>
			<td>Addgene</td>
			<td>60546</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.2-DEST</td>
			<td>Life Technologies</td>
			<td>12489-019</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-DEST</td>
			<td>Addgene</td>
			<td></td>
			<td>http://www.addgene.org/Eric_Campeau/</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-531</td>
			<td></td>
		</tr>
		<tr>
			<td>In-Fusion HD Cloning</td>
			<td>Clontech</td>
			<td>639649</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Clonase</td>
			<td>Life Technologies</td>
			<td>11791</td>
			<td></td>
		</tr>
		<tr>
			<td>Vira Power Lentivirus Packaging</td>
			<td>Life Technologies</td>
			<td>K497500</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE Transfection Reagent</td>
			<td>Roche</td>
			<td>13873800</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Scientific</td>
			<td>155409</td>
			<td>#1.5 glass bottom dishes</td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with phenol red</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM no phenol red</td>
			<td>Life Technologies</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovit&#39;s L-15, no phenol red</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Life Technologies</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral concentrator</td>
			<td>Clontech</td>
			<td>631231</td>
			<td></td>
		</tr>
		<tr>
			<td>Retroviral concentrator</td>
			<td>Clontech</td>
			<td>631455</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>BD Biosciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A11138</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Calbiochem</td>
			<td>345812</td>
			<td>Neomycin</td>
		</tr>
		<tr>
			<td>Doxycyline</td>
			<td>Fisher</td>
			<td>BP2653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Sigma</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm gold particles</td>
			<td>BBI Solutions</td>
			<td>EM.GC100</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Life Technologies</td>
			<td>10977</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn1</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Midiprep kit</td>
			<td>Macherey-Nagel</td>
			<td>740410</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-137</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL&#160; polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>GeneMate</td>
			<td>C-3328-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Life Technologies</td>
			<td>15544</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD Biosciences</td>
			<td>244610</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Fisher</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells</td>
			<td>Life Technologies</td>
			<td>C4040</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

所示的附设-PALM例子是KRAS G12D突变体与RAS相互作用结合CRAF的结构域（RBD）（图1A）。如所讨论的，在RN或RC片段并未标记到的Ras的C-末端，因为它会破坏膜定位的Ras，因此生物活性。这减少了可能的组合，从8至4（图1B）。这四个组合被引入到使用慢病毒感染U2OS细胞。将细胞固定在协议中详述的，并使用PALM设置附设信号进行了评价。细胞与阳性附设信号通过在荧光的突然增?...

Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

photoactivated-localization-microscopy-with-bimolecular-fluorescence

Research

JoVE Journal

Bioengineering

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

9.8K 次观看.  Oregon Health and Science University. Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes. 