我们感谢哈桑 Yardimci 博士和弗朗克斯学院的穆罕默德·塞维姆 Yardimci 博士在单分子实验中的亲切帮助, 丹尼尔博士 Duzdevich 来自哥伦比亚大学格林的实验室, 北京大学宇杰太阳博士和春来牌博士。清华大学进行有益的讨论。这项研究得到了中国国家自然科学基金31371264、31401059、中国科学院跨学科创新小组和来自皇家学会的牛顿高级研究金 (NA140085) 的支持。

1. ARS317 DNA 基质的制备
<ol><li>DNA 基质结构与包装<ol>
<li>用Xho酶消化原生λ DNA;利用含有20个 bp 同源序列的XhoI. 酶在 lambda dna 上的 ARS317, 从萌芽酵母的基因组 dna 中放大543的 bp dna 片段。添加 100 Xho我消化了λ dna 和10的 dna 片段到10µL 同源重组反应系统, 并孵化的反应在37摄氏度为30分钟。</li>
		<li>要包装重组λ DNA, 添加25µL 的 lambda 包装提取物进入重组产品, 并孵化反应在30摄氏度90分钟。然后, 添加额外的25µL 的 lambda 包装萃取物到反应管。继续孵化反应在30°c 90 分钟。</li>
		<li>添加500µL 的无菌稀释缓冲液 (10 毫米三盐酸 (pH 8.3), 100 毫米氯化钠, 10 毫米氯化镁2) 进入反应体系, 并轻轻地将管子翻转几次。加入25µL 氯仿, 轻轻混合, 贮存在4摄氏度。</li>
		<li>添加100µL 的包装噬菌体和100µL 细菌 LE392MP (培养在 0.8-1.0 使用 LB 培养基添加10毫米 MgSO4) 进入一个新的管。孵育37°c 15 分钟。</li>
		<li>添加200µL 的噬菌体细菌混合物成4毫升 oftop 琼脂 (LB 培养基 + 0.7% 琼脂 + 10 毫米 MgSO4, 冷却到48°c)。立即将管子翻转几次, 然后倒入预热的 (37 °c) LB 板上。</li>
		<li>用 PCR 和测序法在37摄氏度的一夜内孵化出平板, 并筛选出λ-ARS317 斑块。</li>
	</ol>
</li>
	<li>从液体裂解物的 DNA 基质纯化11,18<ol>
<li>选择一个牌匾200µL 的无菌 ddH2O 与10毫米氯化镁2和 10 nM CaCl2。与200µL 细菌 LE392MP (在 LB 培养基中培养过夜) 混合, 并在37摄氏度孵化15分钟。</li>
		<li>将噬菌体细菌混合物加入100毫升的 NZCYM (10 克/升新西兰胺, 5 克/升酵母萃取物, 5 克/升氯化钠, 1 克/升 Casamino 水解液, 2 克/升 MgSO4· 7H2O) 培养基和培养 7 h 在37°c。加入250µL 的氯仿, 再摇动10分钟。</li>
		<li>把文化转移到一个200毫升的瓶子里。添加5.8 克氯化钠 (1 米最后浓度), 搅拌溶解的手, 并在冰上孵化30分钟。</li>
		<li>离心机在 1.2万 x g 10 分钟在4°c 去除细胞碎片。将上清液收集到200毫升烧瓶中, 加入 10% PEG8000 (米/V)。搅拌用磁力搅拌装置溶解, 在冰上孵育30分钟或更长时间。</li>
		<li>离心机在 1.2万 x g 10 分钟在4°c 沉淀噬菌体。移除上清。</li>
		<li>并用重悬2毫升的噬菌体稀释缓冲液中沉淀。将其转化为15毫升管, 加入10µL RNase (最终浓度为20µg/毫升) 和40µL DNase (最终浓度为5µg/毫升)。孵育37°c 30 分钟。</li>
		<li>添加2毫升0.3 米三盐酸 (pH 9.0), 100 毫米 EDTA + 1.25% SDS, 15 µL 蛋白酶 K (最终浓度为10µg/毫升)。孵育65°c 10 分钟。</li>
		<li>加入2毫升的预冷醋酸钾 (3 米, pH 4.8), 并在冰上孵化管10分钟。</li>
		<li>离心机在 8000 x g 10 分钟在4°c 去除不溶性的材料。</li>
		<li>将异丙醇的0.7x 体积添加到上清。把管子倒过来翻几次, 室温下孵育2分钟。</li>
		<li>离心机在 8000 x g 10 分钟的 RT, 以沉淀 DNA。移除上清。</li>
		<li>以70% 乙醇为原料, 用 8000 x g 离心法清洗 DNA, 取出上清液。</li>
		<li>洗脱的 dna 使用500µL TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 8.0) 轻轻, 并转移到1.5 毫升管 dna。</li>
		<li>用苯酚提取 DNA 两次: 氯仿离心8000克, 10 分钟。</li>
		<li>沉淀与0.7x 容量异丙醇。将管子轻轻翻转, 然后离心机在 8000 x g 处进行10分钟的混合。</li>
		<li>用70% 乙醇洗一次, 并用重悬200µL。测量 DNA 浓度, 并存储在-20 °c 在12.5 µg 整除数。</li>
	</ol>
</li>
</ol>2. ARS317 DNA Biotinylation
<ol><li>phosphorylate 生物素化寡核苷酸 (5 '-AGGTCGCCGCC-甘醇-生物素-3 ') 补充本机λ DNA 的左端, 添加1µL (100 µM) 的寡核苷酸, 7.5 µL2O, 1 ddH 10x µL 缓冲, 0.5 连接酶µL。孵育反应在37°c 为3小时。</li>
	<li>为 phosphorylate λ-ARS317, 添加12.5 µg 的λ ARS317, 3 µL 10x 连接酶缓冲区, µL T4 1 PNK, ddH2O 到总容积30µL. 孵育反应在37°c 为 3 h。</li>
	<li>用 ARS317 对寡核苷酸进行退火, 添加0.5 µL 磷酸化寡核苷酸, 195 µL ddH2O, µL 10×ligase 缓冲到磷酸化λ-ARS317 管中。轻轻地把管子倒过来, 然后在65摄氏度处孵化5分钟. 关闭块加热器, 让样品冷却到33摄氏度以下的区块。</li>
	<li>结扎寡核苷酸与λ ARS317, 添加1µL T4 DNA 连接酶和0.63 µL 的 ATP (200 毫米)。轻轻地把管子倒过来, 在室温下孵化2小时或4摄氏度。储存在4°c 1 月。 注意: 为了避免分解 DNA, 所有的混合步骤都应该通过轻轻地把管子倒过来, 而不是用吸管混合。</li>
</ol>3. 盖玻片清洁和功能化
<ol><li>盖玻片清洗<ol>
<li>将20盖玻片到4个染色罐 (5 盖玻片/罐), 油脂实验30分钟乙醇, 并冲洗盖玻片与超纯 H2O 3 次。</li>
		<li>油脂实验30分钟, 1 米氢氧化钾 (KOH), 并冲洗盖玻片与超纯 H2O 3 倍。重复乙醇和 KOH 超声波一次。</li>
		<li>用丙酮油脂实验30分钟, 然后彻底冲洗2O (至少3次)。 注: 丙酮必须彻底清除, 清洗与超纯 H2O, 因为它可能会导致爆炸时, 有机溶剂 (如丙酮) 混合与食人鱼解决方案错误14。</li>
		<li>将盖玻片放入食人鱼溶液中 (3:1 混合物 H2,4和 30% H2O2) 和孵化在95°c 为 1 H。 注意: 食人鱼溶液具有高度的能量和侵蚀性, 所以小心使用。当准备食人鱼溶液, 添加50毫升的 h2O2第一个到500毫升玻璃烧杯, 然后添加150毫升的 h2, 所以4慢慢地进入烧杯。</li>
		<li>用超纯 H2O 5 倍冲洗盖玻片。然后用超纯 h2o 彻底清洗每个盖玻片, 使用3烧杯填充超纯2o, 用纸张从盖玻片边缘干燥盖玻片。将盖玻片放入染色罐中, 将盖玻片彻底干燥110摄氏度烤箱30分钟。<ol>
<li>用甲醇冲洗盖玻片, 再将染色罐放入110摄氏度烤箱中, 使盖玻片彻底干燥。 注: 彻底烘干盖玻片是非常重要的, 因为 APTES 在水19的存在下会有许多可能的表面结构。</li>
		</ol>
</li>
	</ol>
</li>
	<li>盖玻片功能化<ol>
<li>将70毫升的硅烷溶液 (93% 甲醇, 5% 醋酸和 2% APTES) 加入每个罐子, 拧紧瓶盖, 在室温下一夜之间离开瓶子。</li>
		<li>清洗和干燥的盖玻片, 如步骤3.1.5 所述, 除了干燥盖玻片彻底使用氮气, 而不是把它们放在烤箱。</li>
		<li>将150毫克的 mPEG (甲氧基聚乙二醇) 和6毫克的生物素 PEG (生物素-聚乙二醇) 分解成1毫升的0.1 米新鲜 NaHCO3 (pH 8.2)。然后离心机在 17, 000 x g 1 分钟, 以消除不溶钉。 注: 新制作的 NaHCO3准备就绪;没有必要调整它的 pH 值。</li>
		<li>把硅烷化盖玻片放在盒子里, 把两个小盖玻片放在硅烷化盖玻片两端的顶部。吸管100µL 的 PEG 溶液在硅烷化盖玻片的中心, 并放置另一个硅烷化盖玻片在顶部。</li>
		<li>添加一些超纯 H2O 在盒子里, 以保持潮湿, 并孵化的盖玻片与 PEG 溶液至少3小时在黑暗中。夜间孵化也能奏效。</li>
		<li>分离盖玻片对, 保持功能面面朝上, 广泛使用超纯 H2O 冲洗盖玻片, 用氮气干燥。</li>
		<li>使用标记笔标记盖玻片在一个角落的功能性一侧, 保持功能性侧面朝上, 把它们放在盒子里, 把盒子存放在真空干燥中1月。</li>
		<li>要储存盖玻片一段较长的时间, 将一个盖玻片成一个50毫升管钻孔, 在其帽上, 然后把管放入一个塑料袋, 并密封袋使用真空封口机。这样, 盖玻片可以储存在-20 °c 约3月。</li>
	</ol>
</li>
</ol>4. 流动单元组件
<ol><li>使用冲床在双面胶带 (30 毫米 x 12 毫米) 的中心处切割15毫米 x 2 毫米通道。</li>
	<li>将双面胶带的纸侧剥离, 并将其粘贴到两个孔的玻璃滑梯上。按下以除去气泡。根据我们的经验, 通过剥离纸侧比双面胶带的塑料面更容易去除气泡。</li>
	<li>使用菱形玻璃抄写器将功能化的盖玻片 (60 毫米 x 24 毫米) 切成四片 (30 毫米 x 12 毫米), 并用氮气去除碎片。记住要保持功能化面朝上。</li>
	<li>剥离双面胶带的塑料面, 并将幻灯片粘贴到功能化的盖玻片上。轻轻按压, 去除盖玻片和胶带之间的气泡。彻底去除气泡可以防止流电池在泵浦时泄漏。</li>
	<li>将入口和出口油管分别插入小孔和大洞中。用环氧树脂固定油管。</li>
	<li>用注射器手动将20µL 的链亲和素 (0.2 毫克/毫升) 泵入流动细胞, 在 RT 中孵育10分钟。然后将10的缓冲液塞进流动单元以取代链亲和素, 并保持室温。</li>
</ol>5. 单分子可视化
<ol><li>利用 532 nm 激光和 640 nm 激光同时激发荧光显微镜试验滑动 #1, 获得红、远红色与 A5 的焦对准。产生分裂光学的双波长图像 (见材料表)。</li>
	<li>将流动细胞放在显微镜上, 将其出口油管连接到一个长油管, 连接10毫升弹簧, 安装在自动输液/提款可编程泵上。 注: 在下面提到的流动单元中, 泵浦缓冲器意味着从流动单元的入口管中提取缓冲液到注射器。</li>
	<li>泵阻塞缓冲器在500µL/分钟进入流单元, 以快速去除空气, 并确保流单元中的缓冲区畅通。然后将阻塞缓冲器在200µL/分钟内泵入流动单元, 翻转出口油管, 彻底去除进气管和流动单元中的气泡。 注: 在使用前至少15分钟, 所有泵入流动单元的缓冲器都应脱气在真空干燥中。</li>
	<li>将生物素化 ARS317 DNA 的0.5 µL 添加到阻塞缓冲器的80µL 中, 并将其泵入流单元25µL/分钟2分钟。然后用200µL 的阻塞缓冲器以50µL/分钟的速率冲洗 ARS317 DNA。</li>
	<li>以50µL/分钟的速率泵送200µL 的绑定缓冲区10 , 以删除流单元格中的阻塞缓冲区。</li>
	<li>添加0.2 µL 链亲和素涂层 Qdot705 (1 µM) 和0.2 µL 的生物素化兽人 (1.2 µM) 成管, 并在室温下孵化5分钟。然后在管子里加入20µL 的结合缓冲器, 放在冰上。兽人-Qdot705的最终浓度约为 10 nM。</li>
	<li>添加2µL 的兽人-Qdot705 (10 nM), 1 µL 的德勤 (100 毫米), 1 µL 的 ATP (200 毫米) 到96µL 的绑定缓冲区。兽人-Qdot705的最终浓度是 0.2 nM。</li>
	<li>泵20µL 0.2 nM 兽人-Qdot705以10µL/分钟的速度进入流动细胞。从过度的兽人-Qdot705中冲出, 使用200µL 的绑定缓冲区, 速度为100µL/分钟。然后将30纳米 SYTOX 橙的粘接缓冲器注入到流动细胞中, 以100µL/分钟的速率染色 DNA 基质。</li>
	<li>利用 405 nm 激光和 532 nm 激光, 激发兽人-Qdot705信号和 SYTOX 橙色染色的 DNA 信号。用四波段带通滤波器 (FF01-446/510/581/703-25) 同时观察100µL/分钟的信号, 用100毫秒的 EM CCD 记录图像。从不同的字段收集20个图像栈。</li>
</ol>6. 数据分析
<ol><li>使用斐济软件裁剪图像的新插件, 这是由我们自己修改的图像插件 (OI_cut_RGBmerge) 开发的美国加州大学旧金山分校的实验室。<ol>
<li>将一叠图像 (512 x 512 像素) 裁剪成两叠图像 (256 x 256 像素)。一个是532纳米激光刺激的结果, 另一个是405纳米激光令人兴奋的结果。</li>
	</ol>
</li>
	<li>使用斐济-图像栈 Z 项目 (平均强度) 处理61序列图像。</li>
	<li>测量 dna 的长度 (ldna) 和距离从兽人-Qdot705 (l兽人) 的网站上绑定 dna 到 dna 的拴端手动。</li>
	<li>用 excel 计算 l兽人/lDNA的结果, 用 R 法分析数据, 然后创建直方图, 拟合高斯分布。</li>
</ol>

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<li>Hughes, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+single-molecule+imaging+reveals+a+direct+interaction+between+UvrC+and+UvrB+on+DNA+tightropes%5BTitle%5D)+AND+%22Nucleic+acids+research%22%5BJournal%5D)">Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.</a> Nucleic acids research. 41 (9), 4901-4912 (2013).</li>
<li>Kad, N. M., Wang, H., Kennedy, G. G., Warshaw, D. M., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collaborative+dynamic+DNA+scanning+by+nucleotide+excision+repair+proteins+investigated+by+single-molecule+imaging+of+quantum-dot-labeled+proteins%5BTitle%5D)+AND+%22Molecular+cell%22%5BJournal%5D)">Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single-molecule imaging of quantum-dot-labeled proteins.</a> Molecular cell. 37 (5), 702-713 (2010).</li>
<li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+Passivation+for+Single-molecule+Protein+Studies%5BTitle%5D)+AND+%22Jove-Journal+of+Visualized+Experiments%22%5BJournal%5D)">Surface Passivation for Single-molecule Protein Studies.</a> Jove-Journal of Visualized Experiments. (86), (2014).</li>
<li>Chen, X. Q., Zhao, E. S., Fu, Y. V. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S2095927317301020">Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts.</a> Science Bulletin. 62 (6), 399-404 (2017).</li>
<li>Yardimci, H., Loveland, A. B., van Oijen, A. M., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+analysis+of+DNA+replication+in+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Single-molecule analysis of DNA replication in Xenopus egg extracts.</a> Methods. 57 (2), 179-186 (2012).</li>
<li>Tanner, N. A., Loparo, J. J., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy%5BTitle%5D)+AND+%22Journal+of+visualized+experiments%3A+JoVE%22%5BJournal%5D)">Visualizing single-molecule DNA replication with fluorescence microscopy.</a> Journal of visualized experiments: JoVE. (32), (2009).</li>
<li>Lockett, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+bacteriophage+%CE%BB+DNA+purification+procedure+suitable+for+the+analysis+of+DNA+from+either+large+or+multiple+small+lysates%5BTitle%5D)+AND+%22Analytical+biochemistry%22%5BJournal%5D)">A bacteriophage λ DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates.</a> Analytical biochemistry. 185 (2), 230-234 (1990).</li>
<li>Smith, E. A., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+prevent+the+loss+of+surface+functionality+derived+from+aminosilanes%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">How to prevent the loss of surface functionality derived from aminosilanes.</a> Langmuir. 24 (21), 12405-12409 (2008).</li>
<li>Kim, Y., Torre, A., Leal, A. A., Finkelstein, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+modification+of+%CE%BB-DNA+substrates+for+single-molecule+studies%5BTitle%5D)+AND+%22Scientific+reports%22%5BJournal%5D)">Efficient modification of λ-DNA substrates for single-molecule studies.</a> Scientific reports. 7 (1), 2071(2017).</li>
</ol>

作者没有什么可透露的。

在这里, 我们提出了一个协议, 以观察 Qdot 标记蛋白和现场特异修饰的λ DNA 的相互作用, 使用 TIRFM 在一个流动细胞。必要的步骤包括对 dna 基质进行特定地点的修饰、dna biotinylation、盖玻片清洁和功能化、流动细胞制备和单分子成像。有两个要点值得注意。首先, 所有与λ DNA 有关的步骤都应该轻轻地操纵, 以减少任何可能的伤害,例如避免反复吹打的混合。第二, 确保盖玻片的清洁程度, 以尽量...

蛋白质和 dna 之间的相互作用对许多复杂的生物过程至关重要, 例如 dna 复制、dna 修复和转录。虽然传统的方法已经揭示了这些过程的性质, 但许多关键机制仍然不清楚。最近, 随着快速发展的单一分子技术, 一些机制已经解决了1,2,3。
单分子荧光显微技术在实时可视化蛋白质-DNA 相互作用中的应用主要取决于荧光检测和荧光探针的发展。对于单个分子研究, 重要的是要用合适的荧光探针来标记感兴趣的蛋白质, 因为荧光检测系统大多是商用的。
荧光蛋白通常用于分子生物学。然而, 低荧光亮度和抗漂白稳定性限制了它在许多单分子检测中的应用。量子点 (Qdots) 是微小的发光纳米粒子4。由于其独特的光学特性, Qdots 的亮度为 10-20 倍, 比广泛使用的有机染料5倍更稳定。此外, Qdots 有一个大的斯托克斯转移 (励磁和发射峰的位置的区别)5。因此, Qdots 可用于长时间观察 (分钟到数小时) 和获取高信噪比的图像, 而不能用于定量测定。
到目前为止, 有两种方法来标记一个目标蛋白与 Qdots 网站-特别是: 标记与 Qdot 共轭的主要或次级抗体6,7,8;或者直接用 Qdots 标记靶蛋白, 这是基于生物素与链亲和素9、10、11、12、13之间的强相互作用。链亲和素涂层的 Qdots 在商业上是可利用的。在最近的研究中, 比拉和 Avi 标记蛋白在体内10的联合表达纯化了高效率的芽酵母中特异的生物素化蛋白。通过以下和优化单分子检测14,15,16,17, 我们观察了 Qdot 标记蛋白和 DNA 在单一分子水平上的相互作用, 使用TIRFM10。
在这里, 我们选择萌芽的酵母起源识别复合体 (兽人), 它可以专门识别和绑定到自主复制序列 (ARS), 作为我们的蛋白质的兴趣。下面的协议提出了一个逐步的过程, 以可视化的互动 Qdot 标记兽人与 ARS 使用 TIRFM。介绍了该部位的制备, 特别是修饰的 dna 基质、dna biotinylation、盖玻片清洗和功能化、流动细胞组装和单分子成像。

<table><tbody><tr><td>Lambda DNA</td><td>New England Biolabs</td><td>N3011</td><td>Store 25 &amp;mu;L aliquots at -20 &amp;ordm;C.</td></tr><tr><td>&lt;em&gt;XhoI&lt;/em&gt; enzyme</td><td>Thermo Fisher Scientific</td><td>FD0694</td><td></td></tr><tr><td>Quick-fusion cloning kit</td><td>Biotool</td><td>B22611</td><td></td></tr><tr><td>MaxPlax Lambda&lt;br/&gt; Packaging Extracts</td><td>Epicentre</td><td>MP5110</td><td>Bacterial strain LE392MP&lt;br/&gt; is included in this package.</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10013092</td><td>Any brand is acceptable.</td></tr><tr><td>Tris</td><td>Amresco</td><td>0497-5KG</td><td></td></tr><tr><td>NaCl</td><td>Beijing Chemical works</td><td>N/A</td><td>Any brand is acceptable.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10012818</td><td></td></tr><tr><td>Chloroform</td><td>Beijing Chemical works</td><td>N/A</td><td>Any brand is acceptable.</td></tr><tr><td>NZ-amine</td><td>Amresco</td><td>J853-250G</td><td></td></tr><tr><td>Casamino acids</td><td>Sigma-Aldrich</td><td>22090-500G</td><td></td></tr><tr><td>PEG8000</td><td>Beyotime</td><td>ST483</td><td></td></tr><tr><td>Magnetic stirring apparatus</td><td>IKA</td><td>KMO2 basic</td><td></td></tr><tr><td>15 mL Eppendorf tube</td><td>Eppendorf</td><td>30122151</td><td>15 mL, sterile, bulk, 500pcs</td></tr><tr><td>Rnase</td><td>SIGMA</td><td>R4875-100MG</td><td></td></tr><tr><td>Dnase</td><td>SIGMA</td><td>D5319-500UG</td><td></td></tr><tr><td>Proteinase K</td><td>Amresco</td><td>0706-100MG</td><td></td></tr><tr><td>Biotinylated primers</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>T4 DNA ligase, T4 DNA Ligase, Reaction Buffer (10x)</td><td>New England Biolabs</td><td>M0202</td><td></td></tr><tr><td>Coverslip</td><td>Thermo Fisher Scientific</td><td>22266882</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10009259</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>306568-100G</td><td></td></tr><tr><td>Acetone</td><td>Thermo Fisher Scientific</td><td>A949-4</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>80120891</td><td>sulfuric acid</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10011218</td><td>30% Hydrogen peroxide</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>322415-2L</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>V900798</td><td></td></tr><tr><td>APTES</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>mPEG&lt;br/&gt; (methoxy-polyethylene glycol)</td><td>Lysan</td><td>mPEG-SVA-5000</td><td></td></tr><tr><td>biotin-PEG&lt;br/&gt; (biotin-polyethylene glycol)</td><td>Lysan</td><td>Biotin-PEG-SVA-5000</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>31437-500G</td><td></td></tr><tr><td>Vacuum desiccator</td><td>Tianjin Branch Billion Lung Experimental Equipment Co., Ltd.</td><td>IPC250-1</td><td></td></tr><tr><td>Vacuum sealer</td><td>MAGIC SEAL</td><td>WP300</td><td></td></tr><tr><td>Diamond-tipped glass scribe</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>70036</td><td></td></tr><tr><td>Glass slide</td><td>Sail Brand</td><td>7101</td><td></td></tr><tr><td>Inlet tubing</td><td>SCI (Scientific Commodties INC.)</td><td>BB31695-PE/2</td><td>inner diameter 0.38 mm; outer diameter 1.09 mm.</td></tr><tr><td>Outlet tubing</td><td>SCI (Scientific Commodties INC.)</td><td>BB31695-PE/4</td><td>inner diameter 0.76 mm; outer diameter 1.22 mm.</td></tr><tr><td>Double-sided tape</td><td>Sigma-Aldrich</td><td>GBL620001-1EA</td><td></td></tr><tr><td>Epoxy</td><td>LEAFTOP</td><td>9005</td><td>five minutes epoxy</td></tr><tr><td>Streptavidin</td><td>Sigma-Aldrich</td><td>S4762</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Infusion/withdrawal&lt;br/&gt; programmable pump</td><td>Harvard apparatus</td><td>70-4504</td><td></td></tr><tr><td>532 nm laser</td><td>Coherent</td><td>Sapphire-532-50</td><td></td></tr><tr><td>640 nm laser</td><td>Coherent</td><td>OBIS-640-100</td><td></td></tr><tr><td>EMCCD Camera</td><td>Andor</td><td>DU-897E-CS0-BV</td><td></td></tr><tr><td>W-View Gemini Imaging&lt;br/&gt; splitting optics</td><td>Hamamatsu photonics K.K.</td><td>A12801-01</td><td></td></tr><tr><td>TIRF illumination system</td><td>Olympus</td><td>IX2-RFAEVA2</td><td></td></tr><tr><td>60&amp;times;TIRF objective</td><td>Olympus</td><td>APON60XOTIRF</td><td></td></tr><tr><td>Quad-edge laser dichroic&lt;br/&gt; beamsplitter</td><td>Semrock</td><td>Di01-R405/488/&lt;br/&gt; 532/635-25x36</td><td></td></tr><tr><td>Quad-band bandpass filter</td><td>Semrock</td><td>FF01-446/510/&lt;br/&gt; 581/703-25</td><td></td></tr><tr><td>Dichroic beamsplitter</td><td>Semrock</td><td>FF649-Di01-25x36</td><td></td></tr><tr><td>Emission filter</td><td>Chroma Technology Corp</td><td>ET585/65m</td><td></td></tr><tr><td>Emission filter</td><td>Chroma Technology Corp</td><td>ET665lp</td><td></td></tr><tr><td>FocalCheck fluorescence, microscope test slide #1</td><td>Thermo Fisher Scientific</td><td>F36909</td><td></td></tr><tr><td>SYTOX Orange</td><td>Thermo Fisher Scientific</td><td>S11368</td><td></td></tr><tr><td>Qdot&lt;sub&gt;705 &lt;/sub&gt;Streptavidin Conjugate</td><td>Thermo Fisher Scientific</td><td>Q10163MP</td><td>Store at 4 &amp;ordm;C, do not freeze.</td></tr><tr><td>ATP</td><td>Amresco</td><td>0220-25G</td><td>Prepare 200 mM ATP solution, using ddH&lt;sub&gt;2&lt;/sub&gt;O, adjust pH to 7.0, and store 10 &amp;mu;L aliquots at -20 &amp;ordm;C.</td></tr><tr><td>DTT</td><td>Amresco</td><td>M109-5G</td><td>Prepare 1 M solution using ddH&lt;sub&gt;2&lt;/sub&gt;O, and store 10 &amp;mu;l aliquots at -20 &amp;ordm;C.</td></tr></tbody></table>

visualizing the interaction between the qdot-labeled protein and site-specifically modified &#955; dna at the single molecule level

荧光显微镜在解剖单一分子水平复杂生物过程的机制方面做出了巨大贡献。在用于研究 DNA-蛋白质相互作用的单分子检测中, 有两个重要的因素需要考虑: 具有足够长度的 dna 基质, 便于观察和用合适的荧光探针对蛋白质进行标记。48.5 kb 的λ dna 是一个很好的候选 dna 基质。量子点 (Qdots) 作为一类荧光探针, 允许长时间观测 (分钟到数小时) 和高质量的图像采集。本文提出了一种在单分子水平上研究 DNA-蛋白质相互作用的协议, 包括制备一个特异修饰的λ dna, 并用链亲和素包覆 Qdots 标记靶蛋白。为了证明概念, 我们选择了在萌芽的酵母中的兽人 (原产地识别复合体) 作为一种感兴趣的蛋白质, 并可视化其与 TIRFM (自主复制序列) 的相互作用。与其他荧光探针相比, Qdots 在单分子研究中具有明显的优越性, 因为它对漂白具有较高的稳定性, 但应注意到该特性限制了其在定量测定中的应用。

为了可视化 Qdot 标记兽人与 ARS 之间的相互作用, 我们首先构造了λ-ARS317 DNA 基底。通过同源重组将含有 ARS317 的 dna 片段集成到XhoI. (33.5 kb) 的原生λ dna 中 (图 1A)。重组产物采用提取物包装, 在 LB 板上培养包装的噬菌体颗粒 (图 1B)。用 PCR 法筛选阳性噬菌体斑块, 并经测序证实 (图 1C)。ARS317 DNA 从液体?...

Watch this Scientific Journal Video about Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified Î» DNA at the Single Molecule Level at JoVE.com

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

在这里, 我们提出了一个协议, 研究 DNA 蛋白相互作用的全内反射荧光显微镜 (TIRFM) 使用一个网站专门修改的λ DNA 基底和量子点标记蛋白。

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

visualizing-interaction-between-qdot-labeled-protein-site

Research

JoVE Journal

Biology

7.5K 次观看.  Chinese Academy of Sciences. 在这里, 我们提出了一个协议, 研究 DNA 蛋白相互作用的全内反射荧光显微镜 (TIRFM) 使用一个网站专门修改的λ DNA 基底和量子点标记蛋白。

视频: Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

科研

生物化学

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫与感染

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

生物学

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

生物分子缩合物中蛋白质相互作用动力学的单分子测量

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

摘要

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生物分子缩合物中蛋白质相互作用动力学的单分子测量

生物分子缩合物中蛋白质相互作用动力学的单分子测量