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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D., 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=Real-time+single-molecule+imaging+reveals+a+direct+interaction+between+UvrC+and+UvrB+on+DNA+tightropes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+dynamic+DNA+scanning+by+nucleotide+excision+repair+proteins+investigated+by+single-molecule+imaging+of+quantum-dot-labeled+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Passivation+for+Single-molecule+Protein+Studies.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-molecule+approach+to+visualize+the+nucleosome+assembly+in+yeast+nucleoplasmic+extracts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+DNA+replication+in+Xenopus+egg+extracts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacteriophage+&#955;+DNA+purification+procedure+suitable+for+the+analysis+of+DNA+from+either+large+or+multiple+small+lysates.">A bacteriophage &#955; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+prevent+the+loss+of+surface+functionality+derived+from+aminosilanes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+modification+of+&#955;-DNA+substrates+for+single-molecule+studies.">Efficient modification of &#955;-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 &#956;L aliquots at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>XhoI 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 
			Packaging Extracts</td>
			<td>Epicentre</td>
			<td>MP5110</td>
			<td>Bacterial strain LE392MP 
			is included in this package.</td>
		</tr>
		<tr>
			<td>MgSO4</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>MgCl2</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>H2SO4</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>80120891</td>
			<td>sulfuric acid</td>
		</tr>
		<tr>
			<td>H2O2</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 
			(methoxy-polyethylene glycol)</td>
			<td>Lysan</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin-PEG 
			(biotin-polyethylene glycol)</td>
			<td>Lysan</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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 
			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 
			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&#215;TIRF objective</td>
			<td>Olympus</td>
			<td>APON60XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-edge laser dichroic 
			beamsplitter</td>
			<td>Semrock</td>
			<td>Di01-R405/488/ 
			532/635-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/ 
			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>Qdot705 Streptavidin Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q10163MP</td>
			<td>Store at 4 &#186;C, do not freeze.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Amresco</td>
			<td>0220-25G</td>
			<td>Prepare 200 mM ATP solution, using ddH2O, adjust pH to 7.0, and store 10 &#956;L aliquots at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Amresco</td>
			<td>M109-5G</td>
			<td>Prepare 1 M solution using ddH2O, and store 10 &#956;l aliquots at -20 &#186;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?...

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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

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...