This work was funded by grant from the BBSRC (grant number BB/H019685/1 and BB/E527663/1) to the University of Exeter.

1，确定的解离常数的近似值（ 即在一个数量级） <ol><li>制备表1中详述的混合物。 </li><li>制备目标配体的库存在可用的最高浓度，以及本然后在6点10倍稀释液。其中一个近似杀敌从现有数据已知的，目的是具有至少两个浓度高于和低于杀敌 。 </li><li>分装18μL混合成8口井的定量PCR板。加入2微升的溶剂中，在第一井。加入2微升的配体的稀释系列（步骤1.2）中的每个成员的一个剩下的七个孔以及每一个。 </li><li>将一个密封的qPCR在板上。为了实现板的良好密封，放置一个手涂布器（见特定试剂的表），在板的中部。平滑向下的密封件一侧，然后重复上的另一半该板。 </li><li>离心反应板在500×g离心2分钟以除去气泡。 </li><li>将板StepOne扩增定量PCR仪。选择“解链曲线”选项，ROX过滤器，并选择快爬坡速率（这提供了一个2分钟的停顿，在25℃，随后的升温到99℃，经40分钟，然后2分钟的停顿）。运行热变性。 在脚本文件执行运行可在网上通过http：注意// <a href="http://www.exeter.ac.uk/biosciences/capsular">www.exeter.ac.uk/biosciences/capsular。</a> </li><li>在仪器运行结束后，点击屏幕上的“分析”按钮。保存结果文件。 </li><li>打开蛋白的热位移的软件。 <ol><li>创建一个新的研究;在属性选项卡，给这一个名字，并在条件选项卡，详细的配体。 </li><li>移动到实验文件选项卡，并导入保存的结果文件（XXX.eds），并设定每口井（FIL模板的内容ES可从作者）。 </li><li>移动分析选项卡，然后按“分析”按钮。 注意：这将分析结果。它可以导出结果作进一步调查与使用Excel导出选项卡。结果导出选项卡划定的格式。最好是在Excel中打开导出的文件，并立即保存为Excel格式。 </li></ol></li><li>检查中的单独溶剂的存在下，蛋白质提供了类似于图1A中所示的结果。接下来，检查结果发现，在“复制”窗格中的熔化温度。确保这说明随着配体浓度明显增加的熔化温度。 请注意：在理想情况下，这将提供一个明确的最大熔解温度（假定蛋白是完全配体结合），以及一个近似杀敌其中熔融温度为配位体的无蛋白和半之间的最大值。 </li></ol> 2，实验装置测定解离常数<ol><li>准备详细的表2作为主结构的混合物。 </li><li>制备的配位体的库存为十五个不同浓度，这将在稀释10倍，在最终的实验。理想情况下，包括浓度高于和低于所估计的杀敌大小的至少两个数量级，并且围绕所估计杀敌的浓度。着眼于七个推定杀敌的一个数量级范围内的点，与在此两边另外四个点;如果有选择，有更多的点，那是饱和值。 注：如果需要的话，它是改变实验条件，使得该配体股是在试验浓度加倍，其中配位体的溶解度的限制是可行的。 </li><li>添加120μL的高手搭配八口井在U形底96孔板，充当水库的主结构方便配药。使用8道移液器免除18微升到PCR板的一列。重复，另外5个栏目，给一共有48口井填在盘上6×8模式。 </li><li>加入20μl的配位体的股票，或溶剂，以便在U形底96孔板分开的孔中。使用8道移液器，吸2微升的八个不同配体的股票（或溶剂）。这些加入的PCR板是充满了预混步骤2.3的一列。重复使用相同的8配体/溶剂股的另外两个列。吸2微升其余八配位体或溶剂的股票，而这些添加在板中的第四列。重复此为另外两个列。这会给一式三份样品的全部16个配体和溶剂的样品。 </li><li>将一个密封的qPCR在板（见步骤1.4）。 </li><li>离心反应板在500×g离心2分钟。 </li><li>将平板中的定量PCR仪。运行使用在步骤1.6中指定的参数的热变性。 </li><li>在仪器运行结束后，点击屏幕上的“分析”按钮。保存结果文件。 </li><li>打开蛋白的热位移的软件。创建一个新的研究;在属性选项卡，给这一个名字，并在条件选项卡，详细的配体。 </li><li>移动到实验文件选项卡，并导入保存的结果文件（XXX.eds），并设置每个内容很好。 注：模板文件可在网上通过http：// <a href="http://www.exeter.ac.uk/biosciences/capsular">www.exeter.ac.uk/biosciences/capsular。</a> </li><li>移动分析选项卡，然后按“分析”按钮。 <ol><li>选择在屏幕的左手侧上的菜单中选择“重复数”标签来显示结果为一式三份。评估的数据的基础上，一式三份如何紧是可靠性。年满D中的一式三份表明重复性差，仔细检查原始数据。 </li><li>同时使用玻尔兹曼或衍生物的方法来评估的熔融温度分析数据。选择“复制结果”选项卡，在“复制结果暗算”，“以剧情：”切换按钮之间的“以旧换新 - 玻耳兹曼”和“以旧换新 - 衍生”。选择给出了样品的更大可重复性的方法。导出的结果作进一步调查与使用Excel导出选项卡。 注意：对于显示多个样品的转换，它几乎总是最好用多个熔体模式的衍生方法。结果导出选项卡划定的格式。最好是在Excel中打开导出的文件，并立即保存为Excel格式。 </li><li>重复实验至少两次，其中包括在一个单独的一天重复，以保证结果的可重复性。如果数据分析（见下文第3步）表明杀敌的值显著不同于原先的估计，相应地改变了配体浓度（见步骤2.2），以保证良好的范围内围绕杀敌值。 </li></ol> 3，数据分析，以确定下解离热变性恒<ol><li>在Excel中创建的配体浓度的表和熔化温度。 </li><li>打开的GraphPad Prism软件，并创建一个XY工作台。输入数据，使用的配体浓度和熔化温度结果在Y列在X列。 注：图中所示的例子是图1B。用公式预装，并使用SPSS统计软件包替代指令的脚本，可在网上通过http：// <a href="http://www.exeter.ac.uk/biosciences/capsular">www.exeter.ac.uk/biosciences/capsular。</a> </li><li>在分析选项卡，选择该选项来改变分析参数S（Ctrl + T键）。输入正确的模型，选择“新建”，“创建新的公式”。将详述于表3为“单点配体结合”的公式。 注意：这些步骤的一个例子示于图1C中 。当使用脚本用方程预装，相关方程可被引导从列表中而不是输入的选择。这个方程的推导在附录中提供。 </li><li>选择“规则的初始值”框中，输入初始值的规则如表3中详细说明。 </li><li>约束的参数P，作为“恒等于”，并输入蛋白的终浓度（在同一个单元作为配位体中给出）。 </li><li>选择确定以进行分析。 注意：这些步骤的一个例子示于图1D。的绘图软件产生一个数字表示的数据，并在适合的模型。吨的例子灰褐色的分析示于代表性数据。 </li></ol> 4，数据拟合，以合作模式为了适应数据合作模式，无论是简单的合作模式，或在两个不同的解离常数定义的模型之间进行选择。第一种方法是优选的负协同的情况下，或者作为初始调查。然而，原则上它是在正协同箱子更好模拟两种不同的解离常数18。在这种情况下，造型可以继续假定任一连续的结合配体，或独立的配体的结合。 <ol><li>遵循相同的初始步骤，在协议3，然而，在步骤3.3，在表3中被列为“简单合作模式”中插入一个方程“的两个配位体序列的结合”，或18“独立的两个配体结合”。 </li><li>选择初始值的相关规则与每个这些方程表3相关联。 </li><li>检查模型与数据的拟合。如果数据符合不佳，可以考虑另一种模式。 注意：这也是很重要的仔细研究的熔融温度，以由蛋白质的热转移软件（步骤2.9）中的数据拟合：有时有必要在这里改变的参数，以获得最佳的结果。另一个要考虑的是数据点的范围是否理想，是否有任何异常点：要么一组数据，在有限杀敌的两侧，或单点异常（特别是在最高配体浓度），可以显著影响结果。 </li><li>重复实验至少两次（见步骤2.12），以确保重现性。 </li></ol> 5，数据拟合为曲线显示在熔融温度二进制班次偶尔，而不是配体分级响应，蛋白质已观察采用二进制响应，其中必然样本显然与未结合的样品中分离。一个例子是，在代表性的结果（ 图4）提供的。在这种情况下，嵌合的熔化温度也不会提供良好的适合杀敌 。 <ol><li>导出由蛋白质的热转移软件输出的原始数据。对于每个温度点，计算平均荧光的零配体和配体的最高浓度。制表从明年这些每个数据点的结果。 注：此处产生的误差小于在嵌合熔融温度的误差。 </li><li>打开SPSS统计软件包。复制的温度下，这两个平均值的数据集，以及数据对于每个实验，在SPSS数据窗口。在变量标签中，设置平均数据集没有配体为“低”，且平均数据集的最高配位体浓度为“高”。 </li><li>可下载的语法文件在网上在http：// <a href="http://www.exeter.ac.uk/biosciences/capsular">www.exeter.ac.uk/biosciences/capsular</a> 。选择“运行→运行所有”。 </li><li>必然结果复制比例到一个新的Excel工作簿，与相关的配体浓度。 </li><li>打开GRAPHPAD软件，并创建一个XY工作台。输入数据，使用的配体浓度和熔化温度结果在Y列在X列。在分析选项卡，选择“改变分析参数”。输入正确的模型，选择“新建”，“创建新的公式”。输入在表3中给出的方程，列为“分析中的熔融温度的二进制移位”。 </li><li>选择“规则的初始值”框中，输入表3中详述的初始值的规则。约束的参数P，如“恒等于”，并输入蛋白质的最终浓度（单位同西甲第二是给出）。 注意：在完成这些方块为在第3协议的例子示于图1C，D。 </li><li>如果有一个良好的配合，其结果可以外推到无限大的配体浓度的预期结果得到改善。从约束在每个配体浓度的比例的模型，检查为配体浓度的最高值的值。如果这是0.99或更大，进一步的分析是不可能的，以改善结果。 </li><li>如果该比例低于0.99，附加的步骤是必需的，以校正最高配体浓度的样品中的未结合蛋白的效果。在步骤5.2中，写的配体结合的，在最高配体浓度点（从步骤5.7）的比例为单元R2（可以使用不同的小区，和R2适当地替换等式表3）。创造最高配体浓度结果的平均值后，一个额外的列。在冷杉T细胞中，复制在表3中列出的方程为“外推到无限大的配体浓度”。这个公式复制到剩余的细胞在此列。 注：此计算中移除未结合的蛋白在最高配体浓度的影响。该配体游离蛋白和最高配体浓度之间的差值乘以绑定在最高配体浓度，以提供在各温度点的充分结合和未结合的蛋白状态之间的预期差的比例的倒数。这种差异被添加或从该未结合状态中减去，得到了充分的配体结合的蛋白质的预期荧光。 </li><li>替换在SPSS数据与此新列最高配体浓度列，重复数据拟合。 注意：步骤5.7 - 5.9，可能需要重复，如果模型显示在绑定在最大配体concentrati的比例进一步显著变化（如果是这样的话，它很可能是理想的，以重复实验与包括较高的配体浓度点）。 </li><li>在情况下，蛋白显示出的二进制移位和显示器合作的行为，该方程在步骤5.5建议应该与那些来自步骤4.1被取代。的“顶”和“底”的参数应当由1和0分别替换。 </li><li>重复实验至少两次（见步骤2.12），以确保重现性。 </li></ol></li></ol>

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The authors declare that they have nothing to disclose.

差示扫描荧​​光显示了其力量的强大和通用的方法表征蛋白质和识别潜在的蛋白配体。在加快蛋白质的稳定性，药物发现（尤其是在欠资金充足的实验室）和结晶10,23-25 ​​有据可查的成就使其成为了化合物的初步筛选一个有吸引力的方法。加入到蛋白的化合物在表观熔融温度-7,9明显的剂量依赖性增加。然而，有过一些尝试，以使用来自这些实验的结果，以确定表观结合常数中排名化合物其亲和力来帮助。这里，我们提出了对系统确定的表观解离常数为蛋白质的配体的存在的方法。 这里给出的结果表明，DSF能迅速，鲁棒提供的解离常数的估计值蛋白质 - 配体结合。所观察到的数据可以用市售的工具来操作，以提供快速测定杀敌的，而不需要做出关于参数的可能值的假设。该方法具有显著优势的是简约中蛋白质和时间需要一些可比方法。这里描述的实验将消耗0.13毫克蛋白每实验（约0.4毫克实验以一式三份重复的）。这与等温滴定量热法（ITC），其中一个实验中，平均40 kDa的蛋白质会消耗同样数量相比，毫不逊色。全套本议定书所需的实验将消耗约4小时，包括准备，为单组实验。再次，这是很可能比方法如ITC或表面等离子共振，这同时有力常常需要相当大的优化以实现最佳的数据相当快。 我们的结果表明，仍然存在着需求仔细研究的原始数据，这些数据的拟合来确定熔融温度和熔融温度数据的拟合，以确定解离常数。第一个挑战是在蛋白融化所产生的原始数据的形状。在某些情况下，该形状可能不是近似于图1A中观察到。常见的问题包括对配体的低温转变结合，高背景荧光，且温度异常多的过渡。低温度的变化被认为具有约束力的一些配体。对于这个方法，最关键的参数是在T m测定的误差，相对于温度的漂移。数据通常可装相当好时三次测量的标准偏差不超过未结合和完全结合的蛋白质之间的熔化温度转变的10％。我们的经验是，如果这样的温度erature位移仅2℃，这可能是足够的，用于安装的数据，如果各个数据点是高度精确的。第二个问题是不寻常的形状的曲线。这些通常是免费的蛋白质和配体结合的形式有所不同，作为配体结合会影响展开的蛋白质模式。在这些情况下，用户必须考虑该数据是否可以适当考虑模型被用来被用​​于确定熔融温度和离解常数。另一个常见的问题是，除了辅因子的蛋白（ 例如，MgCl 2的在我们的例子中与己糖激酶）是必需的，以获得最可靠的数据。我们的经验是，仔细地考虑所有可能的因素在实验中，在服用初期读数必须获得最好的结果的阶段。此外，替代疗法的理论可以揭示这些数据15,17的特点。最后，这是不寻常的某些蛋白质将contain本身暴露的疏水区域，显示高背景荧光。有许多解决这些问题，它已被广泛地审查别处6,9。 特别是，用户必须考虑是否使用玻尔兹曼或衍生物的模型（ 例如， 图4），并在使用的衍生物的情况下，无论是多个熔体必须进行建模。造型的热​​去折叠的两种方法的区别在于玻尔兹曼方法的实验数据拟合至波尔兹曼方程，假设一个正常的S形形状的解折叠曲线。与此相反，该衍生物的方法开出的实验数据的一阶导数在每个点处（ 图1A中的下图），并认为该熔化温度是最高的一次导数的点。 3℃ - 导数方法一般约2返回一个较高的熔融温度。大多数蛋白质会返回一个更一致的结果（ 即，熔融温度为三次重复实验的标准误差是低级）的两种方法之一。这通常是密切相关的蛋白质解折叠曲线的精确形状，并且根据经验确定在每一种情况下，最好的方法是必要的。其中衍生模型的情况下，同样重要的是要考虑多种熔化事件。一些数据清楚地表明依据多个过渡，并且在这些情况下，结果可能是更容易解释，如果这些多个熔化事件建模。在此协议的上下文中，它通常是，加入配位体的可引起蛋白质从具有多个熔化转变到单过渡转移（ 例如，通过稳定的热最脆弱的子域），或反之亦然的情况。因此，我们主张，才考虑这做法将是最好用的原始数据进行检查起来。 继米个人熔化温度odelling，进一步的问题也出现在这些配件在协议部分介绍的车型。当务之急是要仔细检查用对数刻度的配合，以解离常数公式，因为这种分析往往突出了观测数据和模型之间的差异（E·G。， 图3）。而获得的结果通常是健壮的，小心的解释提供了机会，以提取更好的结果，而最含义，从内容通过这些数据提出的一个具体问题是，应该放在这表明协同蛋白质或多个绑定的事件，在DSF的解释。我们，迄今为止，仅观察到了这种现象在蛋白质，预计将有多个特异性结合事件（ 例如，WcbM，一种蛋白质，其最好的同源物是一种多聚体26，和它作为一种多聚体上的大小排阻层析[数据不所示]）。它是不完全清楚，在DSF变性中观察到的负协同表明酶将最终呈现负协同性：更确切地说，这可能是复杂的结合的指示必须使用更大范围的方法来研究更彻底。但这不过建议给我们，这种蛋白质的更广泛的研究，有可能识别有趣的效果。 使用这种方法给出的解离常数的值通常的顺序相同的那些用其它方法，如等温滴定量热法和表面等离子体共振提供。然而，观察到的绝对值经常比用这些方法观察到的高。这是至少部分地一个事实，即解离常数是在含有配体的蛋白质的熔融温度观察到的结果。此杀敌一般比在生理温度更高。该dissociatioÑ​​常数是相关于由方程式反应的温度： <img alt="式（1）" src="/files/ftp_upload/51809/51809eq1.jpg" /> [1] <img alt="式（2）" src="/files/ftp_upload/51809/51809eq2.jpg" /> [2] （其中cθ的标准参考浓度，ΔR G是该反应的吉布斯自由能的变化中，R是摩 ​​尔气体常数，ΔH是在反应的焓变，并且ΔS是在反应的熵变。） 以解离常数的反应在该方法的测量范围通常具有负ΔR G，和因此增加了温度对方程的影响[1]是增加的解离常数。二者构成的吉布斯自由能的ΔH和Δ 的条款（方程[2]）是temperaturë依赖性27，并在解离常数的影响将依赖于这些温度相依性的大小和符号，并且将一定的相互作用有关。因此，它是难以避免的，通过这种方法所确定的解离常数，有时比由在RT操作方法确定更大。温度依赖性的，当然，也对许多其他方法，这些方法倾向于提供的解离常数在温度低于生理温度下一个警告。 DSF的方法的另一个需要注意的是，它是一种标记方法，不像美国国际贸易委员会。使用（SYPRO橙）的荧光标记物是疏水性的，因此，在某些情况下，可以与疏水性配体的蛋白质的结合竞争。因此，很可能在一些情况下，所得到的解离常数将被人为地由于与标签竞争提高。然而，对于不同的配位体的比较中，（主用DSF），差异不太可能充分显著通过亲和力影响化合物的排名。 这种方法的一个潜在缺点是检测限是可以实现的。原则上，它不应该是能够精确地测量用于杀敌即蛋白质浓度低于50％，且在此范围内，即使值很可能是可疑的精度的值。同时检测的范围的这一端的期限可以延长一点通过减少蛋白质和染料的浓度，该仪器的灵敏度会防止进一步降低蛋白质浓度。类似地，灵敏度的上端部将通过所述配体中的溶解度来确定。以获得数学稳健估计中K D，用90％的蛋白质中存在的配体结合的形式，这就要求配位体浓度是近似的获得数据，是最重要的LY 10次杀敌 （假设没有协同）。因此，检测限必然是在有关缓冲区中的配位体的溶解度的十分之一。这意味着，检测方法的限制将在1微米和1至100毫米的范围通常，根据不同的蛋白质和配体。 总之，差示扫描荧​​光，适用范围广的蛋白质的一种通用技术。用这里介绍的方法，可以迅速且廉价地确定为不同的配体的蛋白质的亲和力。这对于在蛋白质纯化和稳定化的应用程序，阐明从宏基因组的酶的功能或特异性的潜力很大，并且在药物发现，特别是在小的实验室。

所有的蛋白质会结合，具有不同的亲和力，从简单的离子等大分子其他分子的多元化。在许多情况下，蛋白质结合的小分子伙伴作为其正常功能的一部分（ 例如，激酶结合ATP）。其它相互作用可能是无关的功能，但是是作为工具（ 例如，小分子，稳定蛋白质，以提高结晶成功，或协助维持在溶液中的蛋白质）实验有用;同时结合于活性位点和蛋白的变构位点可以作为抑制剂，并因此调节酶的活性的小分子。 有多种可用于确定蛋白质的伴侣分子的亲和技术。等温滴定量热1被广泛看作是“黄金标准”，因为它提供了丰富的反应信息，为无标记，并在有限的机会实验rtifacts。然而，尽管在仪表和实验设置自动化的灵敏度最近的改进，它仍然是在蛋白质方面要求相对昂贵的，已经在最佳的低到中等的吞吐量，并且最适合于相互作用的中度至高亲和力（10纳米至100微米杀敌 ）2。其他无标签的方法，如表面等离子体共振或双层干涉3提供更高的吞吐量，并取得了灵敏度探测更小的分子，低至100道尔顿。然而，高通量的仪器，这些方法都是比较昂贵的，是正义的地方会有一个持续的吞吐量相关的项目，因此很可能是无法进入的许多学术实验室。 差示扫描荧光（DSF或thermofluor）于2001年4首次被描述为用于药物开发的方法。在这种实现方法具D，蛋白质是培养用荧光染料（最初萘磺酸的染料被使用），以改变其在结合到蛋白质的疏水区的荧光。蛋白质 - 染料样品，然后加热，并在荧光监测的热量增加。蛋白质的折叠和蛋白的疏水部分暴露，产生了一个特征图案中的荧光作为温度（ 图1A）的函数。该实验可以在小体积中进行的任何商业定量PCR仪，并因此在一个实验中，大量的样品，可同时测试（通常是48，96或384个样品，这取决于仪器型号）。实验通常可以执行在围绕一个小时，提供的样品5的高通量分析的可能性。 该方法的进一步改进已经看到采用的染料具有较好的光谱特性6,7 </SUP&gt;，进行数据分析的通用工具，并建议方案进行初步筛选8,9。方法的应用范围已经扩展，并特别侧重于建立编制和贮藏蛋白10，最佳的条件和确定潜在的具有约束力的合作伙伴，以帮助结晶11。相对高的吞吐量的方法的，在蛋白质的成本相对较低（〜每反应2微克），并适用于学习弱结合分子已取得的DSF基于片段的药物设计的一个非常有价值的工具，特别是在学术环境12-14。 尽管DSF的广泛应用，以研究蛋白质 - 配体相互作用，很少有研究描述的决心，从这些研究中解离常数。然而，这些往往产生描述该蛋白质的展开，具有许多参数的详细方程式必须被装配到稀疏数据或在某些情况下ëstimated 7,15-17。这些方法是特别重要的挑战性的情况下，如紧密结合化合物或蛋白质显示不寻常的过渡。然而，对于许多实验室中，这些详细的分析过于繁琐常规使用。因此，我们建议替代疗法用于不同的场景，并展示如何将这些可用于适应不同的蛋白质 - 配体的相互作用产生的数据。我们的方法使用在StepOne定量PCR仪，为此定制的数据分析软件是可用的;而这加速了数据分析，从其它仪器的结果可以使用以前发表的方法9进行处理，并且在同一下游分析可以被执行。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">StepOne real time PCR instrument</td>
			<td style="width:71px;">Life Technologies</td>
			<td align="right" style="width:119px;">4376357</td>
			<td style="width:167px;">DSF can be performed with many other instruments. The StepOne instrument has very convenient software for data analysis.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Protein thermal shift software v1.0</td>
			<td style="width:71px;">Life Technologies</td>
			<td align="right" style="width:119px;">4466037</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">MicroAmp Fast optical 48-well plates&#160;</td>
			<td style="width:71px;">Life Technologies</td>
			<td align="right">4375816</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Optical sealing tape&#160;</td>
			<td style="width:71px;">Life Technologies</td>
			<td align="right">4375323</td>
			<td>Bio-rad part no. 223-9444 is an alternative supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">U-bottomed 96-well plates</td>
			<td style="width:71px;">Fisher</td>
			<td align="right">11521943</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">SYPRO Orange</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">S6650</td>
			<td>For a smaller volume supplier, use Sigma part no. S5692</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SPSS statistics version 20</td>
			<td style="width:71px;">IBM</td>
			<td style="width:119px;">N/A</td>
			<td>Other statistics packages will provide similar functionality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GraphPad Prism 6.02</td>
			<td style="width:71px;">GraphPad</td>
			<td style="width:119px;">N/A</td>
			<td>Other statistics packages will provide similar functionality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand applicator (PA1)</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">75-3454-4264-6</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hexokinase from Saccharomyces cerevisiae</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H5000</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Glucose</td>
			<td style="width:71px;">Fisher scientific</td>
			<td align="right">10141520</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of protein-ligand interactions using differential scanning fluorimetry

范围广泛的方法是当前可用于确定所述解离的蛋白质之间的恒定和相互作用的小分子。然而，大多数的这些需要访问的专门设备，并且常常需要一定程度的专门知识，以有效地建立可靠的实验和分析数据。差示扫描荧​​光（DSF），正在越来越多地被作为一种可靠的方法用于蛋白质的相互作用的小分子，无论是对生理确定合作伙伴或打的发现初步筛选。这种技术具有需要只适合于定量PCR，并因此合适的仪器是在大多数机构可用PCR仪的优点;一个优秀的一系列协议已经上市;并且有在文献中所述方法的多种用途强先例。以前的工作已提出了计算从DSF数据的解离常数的若干装置，但这些在数学上是苛刻的。这里，我们数字高程模型onstrate用于从DSF的实验数据有中等量估算解离常数的方法。这些数据通常可以被收集，并在一天内进行分析。我们表明模型是如何不同，可以用来拟合，从简单的结合事件收集的数据，并且其中协同结合或独立的结合位点存在。最后，我们提出在标准模型不适用的情况下的数据分析的实例。这些方法被示出具有收集在市售的对照蛋白的数据，并从我们的研究计划的两种蛋白质。总的来说，我们的方法提供了一种简单的方法为研究人员能够迅速获得进一步的洞察使用DSF的蛋白质 - 配体相互作用。

Watch this Scientific Journal Video about Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry at JoVE.com

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

determination-protein-ligand-interactions-using-differential-scanning

Research

JoVE Journal

Biology

61.1K 次观看.  University of Exeter. Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

视频: Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

密度梯度离心法测定微分品质的精子

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

蛋白质 - 配体相互作用采用差示扫描荧光法测定