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1.适体工作股票的制备<ol><li>遵循制造商的指示和（从基准18 5-的Cy5- CCTG GGGGAGTATTGCGGAGGAAGG-3，序列）溶于水的寡核苷酸，达到100μM的最终浓度。 </li><li>通过稀释寡库存至200nM用结合缓冲液制备的适体的工作溶液（20毫摩尔Tris，pH值7.6; 300毫摩尔NaCl; 5mM的MgCl 2的0.01％吐温20）。 </li><li>孵育2分钟，混合物在90℃，让样品立即冷却在冰上，并在室温下使用的样本。 </li></ol> 2.配体稀释系列的研制<ol><li>对于各配体（三磷酸腺苷（ATP），腺苷二磷酸（ADP），腺苷一磷酸（AMP），腺嘌呤，鸟苷三磷酸（GTP），三磷酸胞苷（CTP），脱氧腺苷三磷酸（的dATP），和S-腺苷甲硫氨酸（SAM） ;每个10毫米股票），准备一个16级串行diluti关于在200μl微反应管。 注:5分钟在14000 XG可能有助于除去聚集配股票的离心。低量，推荐使用低结合反应管，以避免分子吸附到所述管的壁。 </li><li>开始的比估计的亲和力高至少50倍的最大浓度和50％在各稀释步骤减少配体的浓度。 注:在控制软件实施的集中查找工具结合模拟数据，并寻找合适的浓度范围稀释系列帮助。 </li><li>填写20μl，以管1.配股票（10毫米）的加10μl适体结合缓冲液到微反应管2至16。 </li><li>转移10微升管1的向管2及通过上下吹打几次适当地混合。转移10微升到下管，并重复此稀释剩余的管子。 </li><li>丢弃10微升过剩从上管。避免以下buffer稀释效应。在管1和管2-16缓冲区必须相同。 </li></ol> 3.最终反应预混液的制备<ol><li>制备具有20微升体积的各个结合反应（10微升适体工作溶液+10微升的各配位体稀释的），以最小化移液误差。只有4微升的体积是苏夫网络cient到FI LL毛细管。 </li><li>添加10微升200nM的适体工作溶液到10微升各配体稀释，并通过上下抽吸数次适当地混合。 </li><li>通过浸渍毛细血管进入样品孵育在室温和音响LL样品为标准毛细管5分钟的样品。潜伏期较长时间可能对一些互动是必要的;然而，5分钟是足够的大多数。触摸仅在侧面的毛细管，而不是在中间的部分，其中该光学测量将采取。 </li><li>放置到毛细血管第Ë毛细盘和启动MST设备。 </li></ol> 4.启动MST设备注:设备提供了两个预安装的软件包，在&#39;控制"软件的实验条件和技术设置&#39;分析"，为产生的数据解释软件。 <ol><li>将毛细管盘成MST设备之前，开始控制软件，并调整通过选择&#39;&#39;"温度控制下拉菜单中的"启用手动温度控制"的总体要求的温度。调整温度至25℃以这种方式。 注:MST仪器就能温度控制在22至45℃。 </li><li>等待温度达到预期的水平，然后将毛细管盘成MST设备。 </li><li>设置LED通道到&#39;红色"对的Cy5染料和调节LED的功率以获得一个FL uorescenc在一个标准的传感器MST设备300到1000荧光单位E信号。 25％的LED功率在该研究中使用。 注:6000至18000荧光单元被推荐用于MST具有高灵敏度的传感器。 </li></ol> 5.毛细管扫描<ol><li>开展毛细管扫描选择了"控制"软件毛细血管位置，点击检查样品的不同质量等方面"开始扫描帽" 出发前的MST测量。 </li><li>检查是否有荧光增强/淬火坚持效果（U形或扁平峰）在软件毛细管扫描。 注:更多关于检测和荧光的处理和贴效果的详细信息可以在讨论中被发现。 </li></ol> 6. MST测量注:在开始测量MST之前，确保排除粘附作用，增强/淬火效果，或移液误差，并确保在毛细管扫描表明FL荧光信号是萨夫音响充分的。有关详细信息，请参阅讨论。 <ol><li>分配从稀释系列中的&#39;控制"的软件的各毛细管位置的配位体浓度考虑混合的适体和配体的稀释步骤。（1:1）。 </li><li>进入配体毛细管＃1的最高浓度（5毫米），选择正确的稀释型（在这里，1:1），点击最高浓度，使用拖动功能来自动分配的毛细血管其余浓度＃2 16。的最低浓度是152.6纳米。 </li><li>输入在控制软件的各个部分中的FL uorescent适体（这里，100纳米）的浓度。 </li><li>使用默认设置，其检测所述FL荧光持续5秒，记录台MST为30秒，并且激光的t灭活后记录再5秒的荧光<html> O监控分子的反扩散。 </li><li>调整激光功率，以20％的在控制软件的各部分。 注意:为了得到最好的信噪比，并避免非特异性的效果，推荐的20-40％的激光功率。在特定情况下，可能需要更高的激光功率，以便获得未结合的和结合的分子的良好分离。 </li><li>保存实验中选择目标文件夹后按&#39;开始MST测量"按钮开始测量MST。 注:.ntp文件将在目标文件夹中生成。使用这种设置，一个测量持续10-15分钟。 </li><li>重复实验过程至少两次以便更准确地确定EC 50值。 注意:为了测试技术再现性，相同的毛细管可以扫描多次（技术性重复）。 </li></ol> 7. MST数据分析核苷酸"&gt;注:分析软件能够在FL Y数据的分析测定时的分析软件绘制相对于配体浓度37 MST时间迹线和在规范化FL荧光（F 范数 ）的变化。 <ol><li>启动MST分析软件（MO.Affinity分析）和加载从目标文件夹中.ntp文件。选择"MST"作为在数据选择菜单中的分析类型。 注:在配体依赖性荧光效应的情况下，最初的荧光可以被选择用于分析。 </li><li>通过拖放和下降，或者按相应的实验运行下面的"+"按钮，各自的技术或生物的run（S）添加到一个新的分析。 </li><li>按各自的实验运行下面的信息按钮以获得关于该实验中，MST痕迹，毛细管扫描，毛细管形状，初始荧光，和漂白率的属性信息。 注意:这些原始数据，可以人因此在分析的后续步骤进行检查。 </li><li>目视检查MST痕迹的聚集和沉淀的效果，可见的颠簸和尖峰。 注:有关检测和聚集效应处理的详细信息，请阅读讨论。 </li><li>目视检查毛细管扫描和毛细管状覆盖吸附效果，扁平形或U形的山峰可见。目视检查毛细管扫描和荧光效应的初始荧光。目视检查漂白效果的漂白率。 </li><li>切换到剂量反应模式，按下相应的按钮改变分析设置为"专家"模式。选择"T-JUMP"作为MST评估策略。 </li><li>选择曲线拟合的"山"的模式。结合参数将自动进行计算。通过选择在"比较结果"菜单的各类型正常化的正常化的数据。导出数据无论是作为一个.xls或.pdf格式。 注:绑定图表下表总结了计算的结合参数。 </li></ol>

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质量控制: 非特异性粘着/样品材料的表面，以及聚集效应的吸附，对亲和数据的质量有巨大影响。但是，只有少数国家的最先进的技术提供准确，快速的选择，监督和避免这些影响。台MST提供了检测和帮助克服这些问题，从而允许技术设置的逐步优化集成质量控制。上粘附和荧光效果的重要信息可以从毛细管扫描和毛细管形状（步骤5.2和7.5）被提取，而聚集/沉淀的效果（步骤7.7）所用的运动轮廓进行监测。这些控制功能使数据质量的不断改善，并表示该协议的关键步骤。 吸附效应和故障诊断检测: 毛细管扫描作为一个ini主要是进行TiAl金属步骤通过在它扫描荧光来确定在托盘支架的毛细血管的位置。每个峰代表一个毛细管，指示用于台MST的测量的起始点。峰形状的目视检查使分子的吸附到玻璃表面上，这是应该开始MST实验之前进行的基本的质量控制的确定。毛细管形状覆盖允许容易地检测扁平，颠簸峰形，甚至类似于一个U形的峰，这表明分子与毛细管（ 图3A，上图）的内玻璃表面的吸附/粘附。不同涂覆毛细管类型（标准，保费，和疏水性）的帮助，以确保分子的毛细血管的溶解度和最小吸附。毛细管应为其适用性的结合实验之前进行测试。洗涤剂如吐温（0.005-0.1％）或普朗尼克F127（0.01-0.1％）马另外，Y防止非特异性吸附作用。优化在此阶段实验的是用于高数据质量（比较图3A，下图）是必不可少的。 荧光效果和故障检测: 在扫描毛细管的峰高度的变化提供对样品特征的信息的另一层。在毛细管荧光随机差异可能，一方面，是由于吹吸错误或，另一方面，从在可能大大增加荧光扫描区域大样本聚集体产生。高吸液精度是强制性的，以避免稀释效应。移液器向上混合所有的解决方案，下进一步提高精度。此外，重要的是要保持在每个毛细管一致缓冲区。策略来处理聚集效应将在后面的段落呈现。 与increa荧光的系统性变化唱配体浓度往往表明配体依赖淬火/增强效果。所述"SD测试"，以便区分来自非特异性荧光损失结合诱导的荧光变化进行的;此测试监视变性条件下的荧光强度。在配体依赖性荧光效应的情况下，变性条件下的荧光强度应该是相同的，独立的滴定剂的浓度。如果在荧光强度的差仍是在这些条件下本，材料或丢失，由于非特异性吸附到管壁或者由于聚集和随后的离心。 为了执行在SD测试中，第一的10微升和10微升的最后配体稀释步骤中的每一个仔细转移至含有10微升2倍的SD混合物（4％SDS和40mM的DTT）的新鲜管中。将样品混合，并且分子变性为在95℃下5分钟。 AFTEř填充样品进入毛细管时，荧光强度进行测定。如果检测到配体依赖性荧光效应，数据可以直接通过从荧光强度的变化推导出的绑定信息进行分析。 的聚集效应和故障诊断检测: 坑坑洼洼，凹凸不平MST时间的痕迹表明聚集和/或沉淀的效果（ 图3B，上图）。非聚集的样品材料显示一个清洁光滑热泳运动轮廓（ 图3B，下图）。样品材料的离心分离在使用前（5-10分钟在14000 xg离心），在加入洗涤剂的（0.005-0.1％吐温-20，0.01％-0.1％的Pluronic F-127，或类似​​的），使用牛血清白蛋白（ &gt; 0.5毫克/毫升），或缓冲液条件（pH和离子强度）的变化，建议以最小化聚集效应，并优化了数据质量。为了获得高质量的数据，它是必不可少的，以尽量减少聚集效应。 数据分析和曲线拟合: 一个热泳运动轮廓被分成不同的阶段，其可以单独地或同时进行检查。 T型跳转介绍了荧光产量随温度变化的突然改变，代表荧光团的内在特性。直接在对高度结合的荧光基团或构象的改变密切结合环境影响T-跳转。较慢的热泳是指在温度场分子的运动，提供在形成的复合物的整体结构的信息。数据分析的默认类型采用了"热泳+ T跳"的设置，利用这两种现象，以确定绑定参数。在情况下，两个相具有相反的方向，建议单独分析阶段。请注意，时间效应和不同物种molec的ULES可能导致在热泳和T-跳转的亲和力差异。分析选项的另一种类型是手动设置，这使得运动轮廓的特定区域的调查。由于聚集或对流扰动可以排除这种方式。为了提高数据质量，游标可能聚集信号之前设置。检查早期热泳通常产生具有更少噪声的数据，因为对流是依赖于时间的过程。这种早期手动设置强烈建议具有高的激光功率（80％）的实验。在选择数据分析设置，集成在分析软件两个装配模型可以应用于以适合结合曲线。从质量作用定律杀敌拟合函数是适合于1:1的结合模式或为具有相同的亲和力的多个结合位点。浓度无关的解离常数K D描述了BOU之间的平衡第二和未绑定状态;因此，一个结合位点的配体的亲合力测定。从希尔方程导出的浓度依赖性EC 50值表示的配位体在该标记的分子的一半在束缚态的有效剂量。希尔配合，应适用于多价结合的机型，特别是如果他们是合作。 集成的质量控制代表了技术，如SPR或ITC MST的一个很大的优势。这些控件允许技术设置的快速和容易的优化，以确保最佳的数据质量。除了时间效率的测量（在15分钟ķd）和自由固定-设置，MST提供缓冲器的自由选择，允许分子间的相互作用的评估，甚至在裂解物和血清5,42,43。由于该分子热泳取决于大小，电荷和分子的水化外壳的事实，没有限制在期间MST的测量测得的相互作用伙伴的大小。动态AF无穷的范围，从下午到毫米，低样品消耗在一起，完成了MST技术的优势。值得一提的是，MST产生基本绑定参数的精确数据，如结合亲和力，化学计量，和热力学。然而，MST不允许k的对测量和K 掉率。此外，一个必须考虑的是，对于大多数的MST实验，荧光修改必须被添加到的相互作用伙伴之一。生成的数据与国家的最先进的技术，如SPR和ITC 32,43-46一致。然而，这些数据是质量控制，所述实验是更快，它们消耗较少的材料。总体而言，MST代表了强大的技术正交于国家的最先进的技术，有一些额外的好处。

分子之间的相互作用是自然的基础。因此，科学家基础和应用研究的众多领域尝试了解不同种类的分子相互作用的基本原则。 MicroScale程序热泳（MST），科学家可以在溶液中进行快速，精确，成本效益和质量控制的分子间相互作用的特征，与缓冲区的自由选择。目前已经有超过1000出版物使用MST，2016年独自一人，描述不同类型的分析，包括图书馆放映，结合事件验证，竞争分析，和实验与多个合作伙伴的结合1-8。在一般情况下，台MST允许古典结合参数，如任何类型的分子相互作用的结合亲和力（1pM至毫米），化学计量，和热力学，研究。 MST的一大优点是研究独立的互动合作伙伴的大小结合事件的能力。即使CHAL目标小核酸适体之间有挑战性相互作用（15-30个核苷酸）和诸如小分子，药物，抗生素，或代谢产物可以定量。 当前国家的最先进的技术来表征适体靶相互作用或者是实验室强烈和高度复杂的或无法量化适配子的小分子的相互作用9,10。表面等离子体共振（SPR）为基础的试验11,12和真正的无标记热量的方法，如温滴定量热（ITC）13-15度洗脱16，平衡渗透17,18，在线探测19，凝胶-移位测定，stopped-溢流FL荧光光谱20,21，荧光各向异性（FA）22,23，单分子FL荧光成像24,25，和生物层干涉测量（BLI）26也无论是不精确或适体-小分子不相容互动。其他principa这些方法升问题是灵敏度低，高样品消耗，固定化，在表面上的质量传递的限制，和/或缓冲的限制。只有少数这些技术提供了聚集和吸附作用的综合控制。 台MST表示一个强大的工具为科学家克服这种限制，研究其它靶的适体和小分子27-29之间的相互作用，以及如蛋白质30-33。该技术依赖于分子通过温度梯度的运动。这种定向运动，被称为"热泳，"取决于大小，电荷，并且该分子34,35的水化外壳。配体分子的结合将直接改变这些参数中的至少一个，从而产生改变热泳迁移率。小尺寸的配体可能没有从绑定尺寸的变化对束缚态方面相当大的影响，但可以拥有博士<html>上水合壳和/或电荷amatic影响。在分子与结合配偶体相互作用后的热泳运动的变化使得基本结合参数2,7,34,36,37的量化。 如在图1A中所描绘的MST装置由红外激光聚焦到使用相同的光学元件作为用于荧光检测的玻璃毛细管内的试样。而激光建立温度梯度（2-6℃的ΔT）可监控的蛋白质经由色氨酸6或荧光标记的相互作用配偶3,8的固有FL荧光的热泳的运动。在空间，ΔT，所产生的温度差会导致在升高的温度下的区域耗尽或分子的积累，这可通过索瑞量化系数音响cient（S T）的 : G"/&gt; C 热表示在被加热区域的浓度，和c 冷是在初始冷区域中的浓度。 如图1B所示 ，一个典型的MST试验结果在MST运动轮廓（时间跟踪），由不同的相位，这可以通过它们各自的时间尺度来分离的。初始荧光在第一5秒中不存在温度梯度的测量来定义的精确开始荧光和检查漂白或p​​hotoenhancement。温度跳跃（T-跳转）表示相，其中热泳动前的荧光变化。荧光这个初始下降取决于uorophore量子产率FL的热依赖性变化。热泳相位如下，其中达到荧光减少（或增加），由于分子直至稳态分布的热泳运动。如在图1B中所示的激光被关闭后，可以观察到的反向TJump和FL uorescent分子伴随背面扩散。为了获得基本的绑定参数，相互作用伙伴不同的摩尔比进行了分析和比较。通常情况下，16个不同的比例进行了研究一个MST实验，而光可见分子被保持恒定，并且与未标记的配体的增加量供给。两个结合配偶之间的相互作用诱导的热泳的变化，并因此在规范化FL荧光，女范数 ，这是因为以下计算: <img alt="方程" src="/files/ftp_upload/55070/55070eq2.jpg" /> ˚F 热和F 冷代表在MST痕迹德科幻奈德时间点的平均荧光FL强度。结合亲和力（K D或EC 50值）可以通过CURV计算易连接（ 图1C）。 总体来看，MST是一个强大的工具来研究任何种类的分子间的相互作用。这份手稿提供了一个协议来表征小分子三磷酸腺苷（ATP 0.5 kDa的）之间的相互作用充满挑战和25-nt的单链DNA短适体DH25.42（7.9 kDa的）。在手稿的过程中，在ATP分子的适体的结合位点被映射下来到腺嘌呤基团的ATP。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Aptamer binding buffer</td>
			<td ></td>
			<td ></td>
			<td >20 mM Tris pH7.6; 300 mM NaCl; 5 mM MgCl2; 0.01%Tween-20</td>
		</tr>
		<tr >
			<td >Fluorescently labeled ATP aptamer</td>
			<td>IDT, Leuven, Belgium</td>
			<td ></td>
			<td>sequence: DH25.42 50-Cy5-CCTGGGGGAGT-ATTGCGGAGGAAGG-3</td>
		</tr>
		<tr >
			<td >ATP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>A2383</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >ADP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>A2754</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >AMP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>A2252</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >Adenine</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>A8626</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >SAM</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>A7007</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >dATP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td >11934511001</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >CTP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td>C1506</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >GTP</td>
			<td>Sigma Aldrich, Germany&#160;</td>
			<td >G8877</td>
			<td>10 mM stock solutions stored at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >Monolith NT.115&#160;</td>
			<td>NanoTemper Technologies, Munich, Germany</td>
			<td >MO-G008</td>
			<td>Blue/Red Channel MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector</td>
		</tr>
		<tr >
			<td >Monolith NT.115 capillaries Standard</td>
			<td>NanoTemper Technologies, Munich, Germany</td>
			<td >MO-K002</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf PCR tubes</td>
			<td >Eppendorf, Germany</td>
			<td >30124537</td>
			<td></td>
		</tr>
		<tr >
			<td >Monolith control software. 2.1.33, pre-installed on the device</td>
			<td>NanoTemper Technologies, Munich, Germany</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >MO.affinity analysis v2.1.1</td>
			<td>NanoTemper Technologies, Munich, Germany</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Kaleidagraph 4.5.2</td>
			<td >Synergy Software</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

mapping the binding site of an aptamer on atp using microscale thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

在这项研究中，MST施加以表征DH25.42的DNA适体18的结合位点上的ATP。相对于其他的研究表征的ATP或ATP的模拟与一个或更多个荧光团38-40随机标记的蛋白质的小分子的相互作用，本研究包括的7.9 kDa的单链DNA适体与一种Cy5的分子标记的版本上的5&#39;端。不同的ATP衍生物和相关分子，都在不同的位置从ATP的不同的，分别用于映射的ATP分子的结合位点。稀释系列（在16步，由每个降低50％的配体浓度）的不同配体的制备和用Cy5标记的适体的恒定量混合。样品在标准的毛细管在25％的LED和20％的激光功率进行分析。运动轮廓（MST时间轨迹）记录和荧光单位，从T-跳转相衍生，被作图配体精矿过程中（比较图1C）。进行曲线拟合施加Hill方程，导致EC 50值。在5个独立的生物重复（4个不同的运营商，2个不同的适体的股票，2种不同的三磷酸腺苷的股票），ATP的适配子相互作用示出的6至13个单元的阴性结合幅度和52.3±5.0微米的平均EC 50值（ 图2A ）。在结合图误差线和"±"的亲和力数据呈现表示的5生物重复（在不同的毛细管5独立测量）的标准偏差。为DH25.42适体与[2,8,5&#39;- 3 H]腺苷和ATP琼脂糖的相互作用以前报道亲和力可比性。与适体的放射性标记的腺苷的相互作用是由离心过滤法进行定量，测定的6±3μm的亲和力（K D）。度洗脱实验与ATP immobil源化的13μM18,41琼脂糖导致的亲和力（K D）。 为了更好地并排侧比较，数据可以标准化为复合的分子的级分（级分结合，FB）使用以下方程: <img alt="方程" src="/files/ftp_upload/55070/55070eq3.jpg" /> 其中， 值（c）是对浓度c测量台MST值， 自由是未结合状态（最低浓度）MST值，以及复合的是完全结合的状态（ 图2B）的台MST值。这归一化是理想的，以不同的配体的比较结果在一个曲线图中， 如图2C所示 。 ADP（63.6±5.9生物学重复μM），AMP的检测AF网络nities（生物重复91.6±9.1μM）和SAM（44.4±3.2微米的生物重复），从ATP的区别在于NU磷酸基团的MBER，意味着该位置没有或仅在上适体（ 图2C和D）的结合行为FL uence较小。在ATP的核糖的C2碳上的OH基团也可以从作为主要结合位点排除在外，因为的dATP也由适体具有稍微降低自动对焦无穷（64.4±6.1生物重复μM）的约束。 ATP的嘌呤基改变为嘧啶基的CTP导致的适体的非结合，这表明了交互这一组的重要性。势必腺嘌呤与141.7±9.4微米的亲和力（在生物重复）的适体，显示出的结合位点必须在ATP分子的这一部分。嘌呤分子GTP和ATP在图2D，它表示对三磷酸腺苷的适体的主要结合位点表示的绿色阴影区域不同。另一项研究中使用的非定量洗脱实验用不同的ATP衍生物从洗脱ATP琼脂糖，显示比较的结果，该研究MST 18的放射性标记的核酸适体。 <img alt="图2" src="/files/ftp_upload/55070/55070fig1.jpg" /> 图1:MicroScale程序热泳 。 （ 一 ）MST技术的技术设置所示。光学重点放在玻璃毛细管的中心位置，从而检测光学可见分子的荧光信号。 IR激光器被用在光学系统的观察窗，以建立温度梯度。在荧光的变化可以被用来监测在一个温度梯度的分子在溶液（B）的MST时间跟踪运动分子廓的热泳运动。初始荧光5秒钟测定，而激光关闭。开关上的激光产生的温度梯度。眼前的T-跳转P的之后，HASE，其中FL uorescent染料减小在热感应其信号收率的热泳运动发生并且观察30秒。激光被关闭后，将分子扩散回来。一个典型的MST实验（C）的结果:（左）的含有荧光分子的相同浓度和未标记配体的浓度增加16个毛细管;在MST时间的痕迹被记录并归其初始荧光。 （右）归FL荧光; ˚F 冷和F 热之间的差作图的配体的浓度。曲线拟合该数据绑定的产量参数，如结合自动对焦无穷的。爱思唯尔，方法28的许可重新印刷;许可证号3890230800113. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55070/55070fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </FONT&gt; <img alt="图2" src="/files/ftp_upload/55070/55070fig2.jpg" /> 图2:MST 数据分析。 （A）中的基线校正的标准化FL荧光ΔF 规范 （‰），从台MST TJump信号导出，绘制在ATP浓度（以μM）。希尔方程（EC 50）应用于曲线拟合。误差条表示从五个生物重复的标准偏差。 （B）的A中示出的数据的各数据组标准化为结合的级分的级分结合的情节，以及这些归一化的数据的平均示于结合曲线图。误差棒表示5生物重复的标准偏差。 （℃）的馏分结合图显示了不同的配体与适体的定量比较（希尔配合）。误差棒代表标准devia二级生物重复化。不同的配体的适体（D）绑定AF网络nities（EC 50）。绿色阴影区域表示在腺嘌呤基团的三磷酸腺苷的适体的结合位点。这个数字是从爱思唯尔，方法28修改;从以往的研究执照号3890230800113.数据集重新分析和研究中扩大。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55070/55070fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55070/55070fig3.jpg" /> 图3:MST实验分析优化。 （A）的蛋白质吸附和粘附的效果可以在毛细管扫描来检测。不规则峰的形状，例如扁平形或U形的峰，表明该样品材料与玻璃表面的粘附。 C挂毛细管类型（标准，保费，或疏水性）可以防止分子吸附，导致在常规峰形。 （B）中的台MST时间迹线还用作质量控制，因为聚集体可以有作为凸块和尖峰来检测。实验条件可以通过提高分子的溶解度（ 例如，包括清洁剂例如Pluronic F-127或变化的pH值或盐浓度）进行优化。离心可帮助去除更大的聚集体。为了确保最佳的数据质量，MST时间的痕迹应类似于给定的例子。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55070/55070fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis at JoVE.com

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

mapping-binding-site-an-aptamer-on-atp-using-microscale

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.5K 次观看.  2bind GmbH. MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

视频: Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

科研

生物化学

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

高亲和力抗体 - 抗原的结合动力学的测定，使用四个生物传感器平台

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...