该协议和算法是在德意志 Forschungsgemeinschaft (DFG 赠款 SFB1009 A09 和 EB177/13-1) 资助的一个项目内开发的。作者感谢芭芭拉 Schedding 和费利克斯 Schmalbein 的技术支持和赖能博士批判地阅读手稿。

1. 库存解决方案
<ol><li>要准备100毫升的 10x tb pH 7.4 溶液, 溶解6.06 克的氯化钠在90毫升的去离子水, 调整 pH 值7.4 与 37% HCl 溶液, 填补体积到100毫升与去离子水, 并过滤解决方案。</li>
	<li>要准备100毫升的1米 HEPES/氢氧化钠, pH 7.4 溶液, 溶解23.83 克的 HEPES 在90毫升的去离子水, 调整 pH 值7.4 与1.5 米氢氧化钠, 填补体积到100毫升与去离子水, 并过滤解决方案。</li>
	<li>要准备100毫升的5米氯化钠溶液, 在90毫升的去离子水中溶解29.2 克氯化钠。用去离子水填充体积到100毫升, 过滤溶液。</li>
	<li>要准备100毫升的1米氯化镁2解决方案, 溶解20.33 克氯化镁2 ·6H2O 在90毫升的去离子水中。用去离子水填充体积到100毫升, 过滤溶液。</li>
	<li>在水中制备5% 热灭活 bsa, 在50毫升管中重2.5 克 bsa (分数 V, pH 7.0), 并将其溶解在45毫升的去离子水中。用去离子水将溶液填入50毫升, 在68摄氏度的水浴中加热溶液45分钟. 在冰浴冷却它并且存放它在-20 °c。</li>
	<li>制备含水25% 戊二醛溶液。 注意: 戊二醛是有害的, 如果吞咽, 有毒的吸入, 并导致烧伤。穿戴防护服、手套和眼睛/脸部防护。</li>
	<li>提高兔抗血清的前述19。根据标准协议20确定抗血清的效价。</li>
	<li>从山羊共轭碱性磷酸酶中制备抗兔免疫球蛋白抗体。</li>
	<li>制备100毫升0.1 米甘氨酸溶液, 在90毫升的去离子水中溶解0.75 克甘氨酸。用1.5 米氢氧化钠溶液将 pH 值调整为 10.4, 用去离子水填充体积到100毫升, 并过滤溶液。</li>
	<li>制备100毫升0.5 米锌 (ii)-醋酸盐溶液, 溶解10.98 克锌 (ii)-醋酸盐·2H2O 在90毫升的去离子水中。用去离子水填充体积到100毫升, 过滤溶液。</li>
	<li>制备100毫升1.5 米氢氧化钠溶液, 在90毫升的去离子水中溶解6.0 克氢氧化钠。用去离子水填充体积到100毫升, 过滤溶液。</li>
</ol>2. 准备缓冲器和工作解决方案
<ol><li>稀释5毫升 10x tb 的 pH 值 7.4, 45 毫升去离子水, 并添加100µL 1 米氯化镁2库存解决方案。保持室温。tb, pH 值 7.4, 2 毫米氯化镁2解决方案是为固定的受体和洗涤微量滴定板。</li>
	<li>稀释10毫升5% 的热灭活 BSA 在水和5毫升 10x tb, pH 7.4 与去离子水到50毫升。添加100µL 1 M 氯化镁2库存解决方案, 并将解决方案进行良好的混合。保持在冰上, 并保存在-20 摄氏度的进一步实验。请注意, 每个滴定曲线都需要2.5 毫升的 tb, 1% BSA, pH 值 7.4, 2 毫米氯化镁2 。</li>
	<li>稀释2.5 毫升的1米 HEPES/氢氧化钠, pH 7.4 和1.5 毫升5米氯化钠溶液到50毫升与去离子水, 添加100µL 1 M 氯化镁2解决方案, 并彻底混合解决方案, 准备50毫升的哈佛商学院, pH 7.4:50 毫米 HEPES/氢氧化钠, 150 毫米氯化钠。</li>
	<li>添加5µL 1M 氯化镁2库存解决方案和2µL 0.5 米锌 (II)-醋酸盐库存溶液5毫升0.1 米甘氨酸溶液, ph 10.4 制备碱性磷酸酶 (AP) 缓冲液 (0.1 米甘氨酸溶液, ph 10.4, 1 毫米氯化镁2, 0.2 毫米锌 (II)-醋酸盐)。</li>
</ol>3. 将受体 (整合素α2A 域) 固定在微量滴定板上
<ol><li>稀释整合素α2A 域的库存解决方案, 在 tb, pH 值 7.4, 2 毫米氯化镁2中, 最终浓度为5µg/毫升。 注: 涂层溶液的体积为一滴定曲线的650µL, 其中包括12井的半面积微量滴定板。</li>
	<li>在一个半区域微量滴定板上填满每一个井, 其中50µL 的5µg/毫升涂层溶液的整合素α2A 域 (野生型或突变体)。至少以重复的方式执行每个滴定行 (在本例中为胎; 请参见图 1中的微量滴定板的布局)。</li>
	<li>用铝箔封住盘子, 或用盖子把它关上。把盘子放在4摄氏度的晚上。</li>
</ol>4. 用 tb、pH 值7.4、2 mM 氯化镁2冲洗两次板材的涂层井
<ol><li>要去除未通过物理吸附固定于塑料表面的可溶性受体分子, 请去除涂层溶液, 并用50µL, pH 7.4, 2 毫米氯化镁2填充每个井。</li>
	<li>卸下清洗液。确保井不变干。因此, 不要把微量滴定板放到纸巾上, 以除去残留的液体。使用多级吸管或多通道吸管快速填满水井。</li>
	<li>重复这个洗涤步骤一次。</li>
</ol>5. 阻止非特异性绑定站点
<ol><li>在 tb、pH 值7.4、2 mM 氯化镁2中, 向每个井中添加50µL 1% BSA 溶液。</li>
	<li>用箔封好井, 或用盖子盖紧。</li>
	<li>在室温下孵化出1小时的水井。 注意: 此协议的任何孵化步骤都可以在摇摆或震动平台上执行。然而, 这不是必需的, 也不会改变实验的结果。</li>
</ol>6. 配体 Rhodocetin 系列稀释排的制备
<ol><li>改变 rhodocetin 的开始浓度和稀释因子的串联稀释, 以获得适当范围的配体浓度和记录一个完整的滴定曲线的最小和最大的信号。在本实验中, 采用 243 nM rhodocetin 的起始浓度和稀释因子2.3。稀释 rhodocetin 的溶液, 达到串联稀释排的最高配体浓度。对于每个滴定曲线的稀释系数为 2.3, 准备115µL 243 nM (即, 152 µg/毫升) rhodocetin 溶液在 1% BSA/tb, pH 7.4, 2 毫米氯化镁2在试管 #1。</li>
	<li>65µL 1% BSA 溶液在 tb, pH 7.4, 2 毫米氯化镁2在10试管, 标记 #2 通过 #11。</li>
	<li>转移50µL 的 rhodocetin 稀释从试管 #1 到试管 #2, 混合两种溶液 (总容积: 115 µL; 稀释系数: 1:2. 3) 由研磨, 然后将50µL 从此混合物转移到试管 #3,等
</li>
	<li>继续此系列稀释, 直到试管 #11。 注: 在步骤 6.1-6.4 中给出的体积足以用于一个滴定曲线。将这些卷乘以复制数。在这种情况下, 执行八滴定曲线 (胎两个α2A 域形式), 并准备以下卷: 920 µL 的 rhodocetin 溶液在最高浓度的试管 #1;520µL 1% BSA 在 TBS, pH 7.4, 2 毫米氯化镁2填写每个试管 #2 #11;和400µL 的传输量从一个管到下一个。</li>
</ol>7. 配体 (Rhodocetin) 在不同浓度下对固定化受体的结合 (整合素α2A 域)
<ol><li>用真空线从微量滴定板井中取出堵塞液。</li>
	<li>立即添加50µL 的 rhodocetin 溶液在 1% bsa/tb, pH 7.4/氯化镁2的试验管 #1 到1列的水井, #2 的试验管的解决方案 2,等添加50µL 1% BSA 在 tb, pH 7。4, 2 毫米氯化镁2 (阻塞和稀释缓冲器) 作为对12列的井的无配体控制 (参见微量滴定板的布局,图1)。</li>
	<li>用箔封好井, 或用盖子盖紧。</li>
	<li>在室温下孵化水井1.5 小时 (约 20-22 摄氏度)。</li>
</ol>8. 用哈佛商学院两次洗井, pH 7.4, 2 毫米氯化镁2
<ol><li>要移除非束缚配体分子, 请移除结合溶液, 并用50µL, pH 7.4, 2 毫米氯化镁2填充每个井。然后, 取出洗涤液。</li>
	<li>当心水井不会变干。因此, 不要把微量滴定板放到纸巾上, 以除去残留的液体。使用多级吸管或多通道吸管快速填满水井。</li>
	<li>重复这个洗涤步骤一次。</li>
</ol>9. 将受体束缚配体与2.5% 戊二醛固定在哈佛商学院, pH 7.4, 2 毫米氯化镁2
<ol><li>通过混合1部分25% 戊二醛溶液和哈佛商学院9部分, pH 7.4, 2 毫米氯化镁2, 准备一个新的2.5% 戊二醛溶液。</li>
	<li>用2.5% 戊二醛溶液的50µL 在哈佛商学院, pH 7.4, 2 毫米氯化镁2中填充微量滴定板的每一个井。室温下孵化微量滴定板10分钟。</li>
</ol>10. 三次洗井, 50 µL/井, pH 7.4, 2 mM 氯化镁2
<ol><li>要去除和停用过量戊二醛, 请取出固定液, 用50µL, pH 7.4, 2 毫米氯化镁2填充每个井。然后取出洗涤液。 注: 将含有戊二醛的固定液从微量滴定板倒入盘中, 并在其被同等容积的 tb、pH 值7.4 后丢弃固定液。</li>
	<li>当心水井不会变干。因此, 不要把微量滴定板放到纸巾上, 以除去残留的液体。使用多级吸管或多通道吸管快速填满水井。</li>
	<li>重复这洗涤步骤两次。</li>
</ol>11. ELISA 法定量检测受体结合配体
<ol><li>增加50µL/井的主要抗体溶液在 1% BSA 在 TBS, pH 7.4, 2 毫米氯化镁2。主要抗体解决方案是对 rhodocetin19, 稀释 1:2, 000 在 1% BSA, pH 7.4, 2 毫米氯化镁2提出的兔抗血清。</li>
	<li>在室温下孵育 75-90 分钟的板材。用50µL (tb)、7.4、2 mM 氯化镁2将板的所有井冲洗三次。在三洗涤步骤中, 不需要将微量滴定板攻到纸巾上。</li>
	<li>增加50µL/井的二次抗体溶液在 1% BSA 在 TBS, pH 7.4, 2 毫米氯化镁2。为此, 稀释二次抗体, 兔免疫球蛋白靶向山羊抗体共轭碱性磷酸酶, 到 1:2, 000 在 1% BSA 在 tb, pH 7.4, 2 毫米氯化镁2。在室温下孵育 75-90 分钟的板材。</li>
	<li>在5毫升的 ap 缓冲器 (0.1 米甘氨酸溶液, pH 10.4, 含1毫米氯化镁2和0.2 毫米锌 (II)-醋酸盐) 中溶解5毫克片剂, 将含有 4-硝基苯基磷酸盐钠的六水合物 (磷酸酶基质) 溶出, 制备 ap 检测溶液。</li>
	<li>在执行下一步之前, 先用50µL/井、7.4、2 mM 氯化镁2冲洗三次板的所有井。在最后一个洗涤步骤后, 把微量滴定板上的纸巾移除所有的液体痕迹。</li>
	<li>添加50µL/井的 AP 检测解决方案的水井的微量滴定板。及时向所有油井添加 AP 检测解决方案, 尽可能同时启动酶转化。因此, 使用多通道吸管。</li>
	<li>在室温下孵化出钢板, 直到溶液中的最高配体浓度变黄。 注: 根据信号强度的不同, 潜伏期可能在5分钟和1小时之间变化。</li>
	<li>加入50µL/井1.5 米氢氧化钠溶液, 停止磷酸酶基质的转化。让盘子站立几分钟, 确保两种溶液无条纹混合。为了保证所有井中相同的潜伏期, 使用多通道吸管, 并添加1.5 米氢氧化钠, 与在步骤11.8 中的 AP 检测基板相同的顺序加入油井。</li>
	<li>用 ELISA 读取器测量每个井405毫微米的光密度 (OD)。</li>
</ol>12. 滴定信号的评估
<ol><li>使用 Excel 打开原始数据表 (OD405nm值)。由于滴定曲线的这些信号值是以行读取的, 因此将这些值转置为一列, 并在另一列中添加配体的浓度作为标签。</li>
	<li>打开 Graphpad 棱镜 5 (版本 5.0)。在主菜单中打开一个新项目文件。在新建数据 & 图形下选择XY格式。为 Y 轴选择输入和绘制每个值的单个点选项。</li>
	<li>将两列、添加配体的浓度和信号值 (OD405nm值) 复制到 excel 文件中, 并将它们分别粘贴到 GraphPad 棱镜的数据表中作为 X 和 Y 值。</li>
	<li>打开 GraphPad 棱镜5的分析子程序, 然后选择XY 分析下的选项非线性回归。选择用户定义的公式, 然后按新建按钮以创建新的公式。</li>
	<li>在表单中键入滴定曲线方程: Y = (Smax Smin) * (x + r + k)-sqrt ((x + r + k) ^ 2-4 * r X))/(2 * r) + Smin + B * X 进入新打开的模板表中, 以 Y 为信号值 S, X 为加配体 L 的浓度, R 为固定化受体的浓度, K 为离解常数, B 为背景斜率。定义适当的约束, 如 K > 0 和 R > 0。 注意: 这个等式是一个不同形式的等式 9的等式。</li>
	<li>通过选择新创建的用户定义的公式来分析数据表的值。用计算得出的近似值 (K、Rt、smax、smin和 B) 打开表, 这些数值显示在软件屏幕左侧的结果部分下。 注: 只有在滴定曲线至少包含5个数据点的情况下, 该软件才通过非线性回归的迭代拟合确定5参数。</li>
	<li>对每组滴定曲线的参数 K、Rt、smax、s最小和 B 进行统计计算, 并将参数与组的特定特征 (突变或化学修饰的配体或受体) 关联。</li>
</ol>

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A., 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=Dramatic+and+concerted+conformational+changes+enable+rhodocetin+to+block+&#945;2&#946;1+integrin+selectively.">Dramatic and concerted conformational changes enable rhodocetin to block &#945;2&#946;1 integrin selectively.</a> PLoS Biol. 15 (7), e2001492 (2017).</li><li>Bracht, T., Figueiredo de Rezende, F., Stetefeld, J., Sorokin, L. M., Eble, J. 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作者没有什么可透露的。

滴定 ELISA 是一种多功能的测试系统, 用于测定受体配体相互作用的离解。由于滴定 ELISA 绕过了有效分离游离和束缚配体的必要性, 并定量分析了它们的浓度, 大量的研究和出版物使用滴定 ELISAs 而不是记录绑定曲线.此外, 滴定 ELISAs 是容易执行和要求相当低数量的受体和配体。为了准确分析离解常数, 必须使用纯制剂和受体和配体。在受体制剂中污染蛋白质与微量滴定板塑料表面吸附部位的受体竞?...

离解常数 K 是描述受体 (R) 对配体 (L) 亲和性的关键参数。以质量作用定律为基础, 定义了受体配体复合 RL 离解受体 R 和配体 L 的平衡:
<img alt="Equation 1 " src="/files/ftp_upload/57334/57334eq1.jpg">            等式1
索引f指示受体和配体的自由/未绑定状态。受体配体复合物的浓度, RL, 是相同的受体绑定配体 Lb的浓度。当受体 rt的总浓度是自由受体 rf和配体绑定受体 rb = Lb的总和时, 离解常数也可以写成:
<img alt="Equation 2 " src="/files/ftp_upload/57334/57334eq2.jpg">        等式2
因此, 饱和屈服 Y, 定义为绑定配体 Lb的分数与受体 Rt的总浓度的关系,
<img alt="Equation 3 " src="/files/ftp_upload/57334/57334eq3.jpg">        等式3
取决于游离配体 Lf的浓度:
<img alt="Equation 4 " src="/files/ftp_upload/57334/57334eq4.jpg">        等式4
这种双曲关系描述了受体配体相互作用的结合曲线, 其图形显示了束缚配体 lb作为游离配体 lf浓度的函数的浓度。从结合曲线上可以导出游离配体在半最大饱和屈服下的浓度。此外, 还建立了进行线性化绑定曲线的不同算法, 如Klotz1、2或根据斯卡查德或哈内斯进行的转换 (由Bisswanger3 检查) 的双对等图。然而, 所有的算法都存在一个问题, 即饱和屈服的最大值在结合曲线上的自由配体的高浓度下渐近逼近, 必须在图形预评价中得到估计, 因此容易出错。
另外, 结合曲线的确定要求在束缚平衡期间定量的自由和束缚配体。为此, 游离配体必须与受体结合的配体分离并量化。因此, 配体和受体必须在其性质上有所不同, 如非蛋白配体, 而不是蛋白质受体。如果两个具有约束力的合作伙伴都是蛋白质, 它们就必须在大小、电荷或其他分子特征上区分开来。然而, 小尺度结合方法中配体浓度的量化是一项艰巨的任务。对配体进行放射性标记往往是检测束缚配体低浓度的必要条件, 尤其是在大量的受体无法获得或负担得起的情况下。此外, 受体结合的配体可以在隔离期间和之后, 以不可忽略的方式分离。因此, 复杂的方法, 如平衡凝胶过滤4, 毛细管电泳5和脉冲蛋白质分解6, 需要量化受体绑定配体, 并将其与游离配体分离。
与这些结合化验相比, 滴定实验不需要定量分离的束缚和游离配体。为此, 在恒定浓度的受体滴定不同浓度的添加配体。通过绑定到受体, 绑定配体有一个生物物理性质, 它区别于游离配体, 是可测量的,例如, 光度, 荧光, 或抗体检测。因此, 信号 S, 与饱和率 Y 和因而也到受体束缚配体 (lb) 的浓度成正比, 被检测为添加配体 (lt) 总浓度的函数。两个参数, 信号 S 和添加配体的总浓度比束缚和游离配体的浓度直接和容易的方式量化。特别是通过酶联免疫吸附试验 (ELISA) 检测受体结合配体, 使试样体积减少到100µL 以下, 以及多井微量滴定板中几种配体浓度的平行测量。在滴定 ELISA 中, 受体被物理吸附到微量滴定板上, 在同一浓度和滴定与可溶性配体。该受体主要通过疏水性吸附固定于塑料表面。固定化受体的表面浓度与受体在非线性关系中的涂层浓度相关, 可能根据 Langmuir´s 吸附等温线7。除了吸附受体分子的总数量外, 它们的活性状态也是滴定测定的另一个重要参数。只有固定的受体保留配体结合活动, 是相关的滴定分析和最终贡献的总浓度的活性受体 Rt滴定法, 不能直接确定。
非固定化受体覆盖的塑料表面上的部位容易吸附其他蛋白质, 如配体。对这种塑料表面的配体进行物理吸附会导致类似的信号作为受体束缚配体, 但以非特异性的方式。为了减少这种非特异性信号, 尚未涂上蛋白质的微量滴定板的塑料表面部位会被牛血清白蛋白 (BSA) 阻断。然而, 对于某些受体配体滴定法, 可以观察非特异性的背景信号。然后, 建议使用其他阻挡剂, 如0.2% 明胶或0.04% 吐温20的溶液。
与受体结合后, 游离配体被两个洗涤步骤除去。束缚配体仍然与受体, 这是固定在塑料表面的微量滴定井, 并选择性加强化学固定。对于随以戊二醛的束缚配体与固定化受体的共价键交联, HEPES 中的缓冲物质三被取代, 而不改变配体结合。HEPES, 与三个不同, 不灭活戊二醛。戊二醛共价交联固定与受体的束缚配体, 防止其离解在随后的洗涤和孵化步骤。因此, 受体配体相互作用是化学冻结, 并保证一个滴定曲线, 不受随后的洗涤和孵化步骤的影响。然而, 戊二醛固定可能化学修饰的配体和受体, 这样的方式, 他们的相互作用减少或废除。此外, 修饰配体内的表位可能改变检测抗体的结合亲和性, 特别是当单克隆抗体用于量化绑定配体。尽管这两种戊二醛固定的不良影响都发生在这种滴定法中, 但对戊二醛的检测灵敏度必须在滴定实验之前对每种受体配体相互作用进行测试。固定后, 过量戊二醛被去除三洗涤步骤与三含缓冲。三钝化剩余醛类, 这可能 nonspecifically 反应与检测抗体在随后的步骤。
结合酶联抗体定量分析了束缚配体的数量, 提供了光度 ELISA 信号。这是绘制的与总配体浓度 Lt添加到每个井。尽管它更容易获得, 滴定曲线不是一个双曲函数与结合曲线的对比。此外, 如何从滴定曲线计算离解常数 K 也不清楚。虽然 Stockell8和海恩和 Weischet9独立地报告了进行线性化 spectroscopically 获得滴定曲线的算法, 但由于它们估计的最大信号值的不确定性, 它们的数量很短。高浓度添加配体的饱和屈服方法。
本文介绍了一种滴定法和非线性回归算法, 从滴定曲线上推导出受体配体相互作用的离解常数 K。这一协议的例子, 以相互作用的胶原结合 a 域的整合素α2β1与蛇毒衍生抑制剂。整合是细胞黏附分子, 它调解细胞的锚地到周围细胞外基质或底层基底膜10,11。此外, 整合在细胞和细胞外基质之间传递重要信号, 通过招募额外的信号分子和形成新的细胞器, adhesomes, 在细胞基质相互作用12,13,14. 胶原蛋白是α2β1整合素的配体, 是人体最丰富的蛋白质, 是结缔组织15的关键支架组件。α2β1整合素与胶原蛋白之间的相互作用是由α2亚单位的 A 域介导的。整合素α2A 域含有价阳离子, 它是胶原结合所必需的, 它的结构稳定。野生型的形式以及α2A 领域的突变体, 如 Y216 被取代为甘氨酸的表面暴露的残留物, 可以很容易地在细菌表达系统中产生 recombinantly, 并通过其寡核苷酸与 NiNTA 分离。superflow 柱与随后透析对三缓冲盐水 (tb; 50 毫米三/HCl, pH 值 7.4, 150 毫米氯化钠) 包含2毫米氯化镁216。它们的浓度是用二辛可宁酸测定法 (纯度) 测定的, 它们的含量由常规的 SDS 页进行测试, 并沾上了考马斯--灿烂的蓝色 R250。
α2β1整合素与胶原蛋白的相互作用是由 rhodocetin 的蛇毒成分 (Calloselasma rhodostoma)16,17) 的结合所阻断的。作为可溶性配体在这个滴定酶联免疫吸附, rhodocetin 是从粗毒液纯化, 如前所述16。它被溶化在 HEPES 缓冲的盐水 (哈佛商学院; 10 毫米 HEPES/氢氧化钠, pH 值 7.4, 150 毫米氯化钠) 并且被存放被冻结在-20 °c。它的浓度是由鉴定者决定的, 其纯度由 SDS 页面证明。作为拮抗剂, rhodocetin 不仅阻断了整合素α2β1 A 域的胶原蛋白结合, 而且稳定了整合体的非活性构象, 从而防止胶原蛋白进入细胞或血小板18。rhodocetin 与受体靶的分离常数的测定具有重要的生物医学意义, 从而解开其分子机制和药用电位如, 作为抗血栓剂。为此, 介绍了一种滴定 ELISA 法, 包括其评价, 适用于几乎任何受体配体相互作用的1:1 相互作用化学计量。

<table>
	<tbody>
		<tr>
			<td>TRIS</td>
			<td>neoFrox</td>
			<td>1125KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Applichem</td>
			<td>1,316,591,214</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Merck</td>
			<td>172571</td>
			<td></td>
		</tr>
		<tr>
			<td>integrin a2A, wild-type and mutant, recombinant</td>
			<td></td>
			<td>isolated in author&#39;s lab</td>
			<td></td>
		</tr>
		<tr>
			<td>NiNTA superflow column&#160;</td>
			<td>Qiagen, Germany</td>
			<td>30821</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie-Brilliant Blue R250</td>
			<td>Serva</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>bicinchoninic acid assay (BCA), protein concentration determination kit</td>
			<td>Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumine (BSA), fraction V</td>
			<td>Applichem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>25 % solution of glutaraldehyde</td>
			<td>Merck</td>
			<td>354400</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit immunglobulin-antibodies from goat, conjugated with alkaline phosphatase</td>
			<td>Sigma-Aldrich</td>
			<td>A9919</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Applichem</td>
			<td>A1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Zn(II)-acetate</td>
			<td>Applichem</td>
			<td>A4324</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Applichem</td>
			<td>A1551</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase substrate tablet (5 mg)</td>
			<td>Sigma-Aldrich</td>
			<td>S0942</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar half-area microtiter plate</td>
			<td>Thermo Scientific</td>
			<td>Corning 3690</td>
			<td></td>
		</tr>
		<tr>
			<td>micro reaction tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate ELISA reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
	</tbody>
</table>

titration elisa as a method to determine the dissociation constant of receptor ligand interaction

离解常数描述二个伙伴之间的相互作用在束缚平衡并且是衡量他们的亲和力。它是比较不同配体的关键参数,例如, 竞争性抑制剂, 蛋白质亚型和突变体, 其结合强度的结合的合作伙伴。离解常数通过绘制绑定vs自由配体的浓度作为绑定曲线来确定。相反, 滴定曲线, 其中一个比例的浓度的绑定配体被绘制反对总浓度的附加配体, 更容易记录。该信号可以 spectroscopically 和酶联免疫吸附试验 (ELISA) 检测。这在一项滴定 ELISA 的协议中得到了证明, 它测量了蛇毒衍生 rhodocetin 对其固定化目标域α2β1整合素的约束力。滴定 ELISAs 是多才多艺和广泛使用。任何一对相互作用的蛋白质可以作为固定受体和可溶性配体, 只要两种蛋白质是纯的, 他们的浓度是已知的。到目前为止的困难是确定从滴定曲线的离解常数。本文介绍了滴定曲线的数学函数。在没有任何容易出错的饱和率图形估计的情况下, 该算法可以通过非线性回归的数学评价直接处理原始数据 (不同浓度的附加配体的信号强度)。这样, 可以同时记录几种滴定曲线, 转化为一组特征参数, 其中包括解离常数和结合主动受体的浓度, 并对其进行统计学评价。结合该算法, 滴定 ELISAs 获得直接呈现离解常数的优点。因此, 将来可能会更有效地使用这些方法。

在 ELISA 被开发以后, 被转换的碱性磷酸酶基体的黄色颜色, 对位-复硝酚表明, 束缚的 rhodocetin 配体的数量减少以增加的 rhodocetin 的浓度从列1到 11 (图 1)。12栏无 rhodocetin 井中的无色井显示了低背景信号。
光度量化在405毫微米提供数据, 可以直接地被哺养入分析软件, 并且通过使用一个非线性回归和<st...

Watch this Scientific Journal Video about Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction at JoVE.com

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

描述了执行滴定 ELISA 的详细协议。此外, 提出了一种新的评价滴定 ELISAs 的方法, 并获得了可溶性配体对微量滴定板固定化受体的离解常数。

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

titration-elisa-as-method-to-determine-dissociation-constant-receptor

Research

JoVE Journal

Biochemistry

19.6K 次观看.  University of Münster. 描述了执行滴定 ELISA 的详细协议。此外, 提出了一种新的评价滴定 ELISAs 的方法, 并获得了可溶性配体对微量滴定板固定化受体的离解常数。

视频: Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

细胞膜亲和色谱柱鉴定与固定化托肌球蛋白激酶受体B相互作用的特化植物代谢物

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...