Name: measuring protein binding to f-actin by co-sedimentation
Uploaded: 2017-05-18T12:30:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

这项工作得到了国家卫生研究院授予的HL127711给AVK的支持。

准备材料<ol><li>净化感兴趣的蛋白质（见第2部分）。 </li><li>准备或购买G-肌动蛋白。 注意:G-肌动蛋白可以从多个来源分离1 ;或者，可以购买。重组的G-肌动蛋白（在5mM Tris pH 8.0,0.2mM CaCl 2，0.2mM ATP（三磷酸腺苷）和0.5mM二硫苏糖醇（DTT）中）应该被快速冷冻，储存在-80℃和&gt; 10mg / mL在小（10-20μL）等分试样中，并在使用前解冻。不要将G-肌动蛋白等分试样重新冷冻。 </li><li>准备或购买对照蛋白，如BSA（参见步骤4.4）。 </li><li>制备10倍聚合缓冲液（200mM咪唑pH 7.0,1M KCl，20mM MgCl 2，5mM ATP和10mM EGTA（乙二醇 - 双（β-氨基乙基醚）-N，N，N&#39;四乙酸））。制成10x原料，加入ATP后调节pH值，必要时。等分（25μL是有用的体积）并存储在-80＆＃176℃。 </li><li> 制备10x反应缓冲液（200mM咪唑pH 7.0,1.5M NaCl，20mM MgCl 2，5mM ATP和10mM EGTA）。制成10x原料，加入ATP后调节pH值，必要时。等分试样（50-100μL是有用的体积），并储存在-80℃。 注意:反应缓冲液的组成是柔性的，可能需要调整以减少背景沉淀，限制非特异性结合和/或改善结合（步骤1.5.1-1.5.3）。 <ol><li>调整反应缓冲液的pH值在6和8之间以优化蛋白质的稳定性。在较低或较高的pH值下，用适当的缓冲液代替咪唑。 注意:使用pH 7.0作为起点，除非蛋白质需要更低或更高的pH值才能稳定。不要使用pH低于6.0或高于8.0的缓冲液，因为这可能会扰乱肌动蛋白。推荐的缓冲液（终浓度和最佳pH列出）包括:20mM MOPS（3-（ N-吗啉代）丙磺酸），pH= 6.5; 20mM咪唑，pH = 7.0; 10mM HEPES（4-（2-羟乙基）哌嗪-1-乙磺酸），pH = 7.5;和20mM Tris，pH = 8.0。 </li><li>根据测定的需要，改变反应缓冲液的盐浓度。 注意:肌动蛋白是一种酸性蛋白质，几乎所有的肌动蛋白结合蛋白在一定程度上依赖于与肌动蛋白相关的静电相互作用。因此，在大多数情况下，增加盐浓度会降低肌动蛋白的结合。反应缓冲液使用生理盐水（150 mM NaCl，工作浓度），这是推荐的起点。如果需要，盐浓度可以降低（ 例如， 100mM）以促进结合或增加以限制结合。 </li><li>不要改变反应缓冲液中MgCl 2 ，ATP或EGTA的浓度，除非有特定的原因。 </li></ol></li></ol> 2.准备测试蛋白质用于测定<ol><li> Preparea高纯度蛋白质使用液相色谱法获得最佳效果7 。 注意:如果使用重组蛋白，应通过蛋白酶切割靶蛋白来去除大量蛋白质标签，如谷胱甘肽-S-转移酶（GST），因为它们会干扰结合。 GST还引起融合蛋白的同二聚化，这可以人为增加肌动蛋白结合的亲和力。 </li><li>通过测量280nm处的吸光度来确定蛋白质浓度。除以消光系数;可以使用序列分析或在线工具从蛋白质序列计算消光系数。或者，使用Bradford或BCA（二喹啉酸）方法测定蛋白质浓度。 注意:对于初始实验，20-40μM的50-100μL蛋白质通常是足够的。这将允许分析低微摩尔范围内的结合，这是大多数肌动蛋白结合蛋白的有用起点。更大的一个并且通常需要较高浓度的蛋白质以产生结合曲线以计算亲和力（参见第5节）。 </li><li>在使用之前，将蛋白质硬转化（在4℃下50,000-100,000xg 10分钟）以除去不溶性蛋白质的聚集体。如果是溶解度，离心后重新测量蛋白质浓度（步骤2.2）。 </li></ol> 3.准备F-肌动蛋白<ol><li>从-80°C冰箱中取出G-肌动蛋白等分试样，并迅速解冻。 </li><li>将10倍聚合缓冲液加入到G-肌动蛋白中至终浓度为1倍。确保1x聚合缓冲液中的G-肌动蛋白浓度至少为10-20μM，远高于临界浓度。在室温（RT）孵育1小时以使肌动蛋白聚合。 </li><li>聚合后，将F-肌动蛋白储存在4℃的溶液中，保持稳定数周。在储存后再次使用F-肌动蛋白后，轻轻倒转或者将管子轻轻松开以确保所有肌动蛋白溶解并均匀分布在溶液中。 注:（可选）添加鬼笔环肽以达到摩尔比为1:1的G-肌动蛋白:鬼笔环肽。在室温下孵育30分钟，以使鬼笔环肽与F-肌动蛋白结合。鬼笔环肽稳定F-肌动蛋白并完成两件事情:（i）减少在离心过程中不沉淀的肌动蛋白的量，（ii）它允许将F-肌动蛋白稀释到临界浓度（〜0.5μM）以下如果改变F-肌动蛋白的量以产生结合曲线（参见关于测量亲和力的第5节），则是必需的。 </li></ol>造血细胞测定 - 基本方案注意:第4节中描述的基本方案用于确定感兴趣的蛋白质是否与F-肌动蛋白共沉淀。一旦建立了与F-肌动蛋白的结合，则可以按照第5节中描述的方案来测量该相互作用的亲和力。 <ol><li>通过将10x原料稀释至1x并加入DTT至终浓度为1 mM，将反应缓冲液制备成使用日。 注:（可选）在反应缓冲液中加入最终工作浓度为0.02％的聚多卡醇。波立多醇是减少非特异性背景结合并有助于防止疏水性蛋白质粘附到超速离心管的表面活性剂。 </li><li>在超离心管中，将1x目的浓度的目标蛋白稀释至1x反应缓冲液中。保持样品体积低（40-60μL），以避免使用大量的蛋白质，通过使用体积小的超离心管（ 例如 ，每个保持0.2 mL的7 x 20 mm管）。 注意:由于许多肌动蛋白结合蛋白对微摩尔范围内的F-肌动蛋白具有亲和力，所以建议以2和10μM测试感兴趣的蛋白质。如果在10μM未观察到结合，则在较高浓度下不可能观察到结合。如果添加的蛋白质占最终反应体积的10-20％以上，可能需要在进行实验之前将蛋白质透析到反应缓冲液中。 </li><li>将F-肌动蛋白加入到所需的最终浓度。 注意:2μM是初始实验的有用浓度，因为它远远高于临界浓度，从而保持肌动蛋白处于丝状状态。当通过SDS-PAGE（步骤4.10）分析时，它将产生可见的颗粒。 </li><li> 在超速离心管中准备以下控制以使测定信息丰富。 <ol><li>准备含有没有F-肌动蛋白的目的蛋白质的样品。确保这些样品中的蛋白质浓度与"加F-肌动蛋白"样品中的浓度相符。 注意:这些样品将确定在没有F-肌动蛋白的情况下聚集或粘附在超离心管侧面的蛋白质的量。 </li><li>准备阴性对照样品以与用于感兴趣的蛋白质相同或相似的浓度。使用不含F-肌动蛋白的不结合F-肌动蛋白的对照蛋白。 注意:这是一个重要的控制，因为即使蛋白质不结合F-肌动蛋白，蛋白质可以在肌动蛋白丝和F-肌动蛋白沉淀中被"捕获"。捕获的量可以根据F-肌动蛋白源，缓冲液条件等而变化。因此，该控制应包括在所有实验中。理想情况下，对照蛋白应具有与感兴趣的蛋白质相似的分子量（ 例如 ，对于αE-连环蛋白（〜100kDa），BSA（66kDa）是适当的对照）。商业上可获得的凝胶过滤标准品制备出优异的对照蛋白质，因为它们涵盖一定范围的尺寸，并且倾向于不含聚集体。 </li><li>任选地，制备含有和不含F-肌动蛋白的含有结合F-肌动蛋白的蛋白质的阳性对照样品。确保浓度类似于e蛋白质。 注意:该控制有助于证明实验条件（ 例如，制备的F-肌动蛋白，反应缓冲液和离心）允许F-肌动蛋白结合。由于造粒测定法不能检测到弱F-肌动蛋白相互作用（参见讨论），建议已知的F-肌动蛋白结合蛋白对F-肌动蛋白具有中等至弱的亲和力（ 即，在低微摩尔范围内）。纯化的F-肌动蛋白结合蛋白是可商购的。 </li></ol></li><li>在室温下孵育所有样品30分钟。 注意:假设感兴趣的蛋白质是稳定的，尽管可能不必要，培养时间更长。如果感兴趣的蛋白质在RT时不稳定，则样品可以在4℃下孵育。在这种情况下，可能需要较长的孵育时间。 </li><li>将样品装入离心机转子。将管置于转子内，以​​帮助离心后将沉淀物重新悬浮。为此，请标记所有离心管（ 例如，带有样品编号），并将转子中的所有管放置在相同的位置（ 例如，数字朝外）。 </li><li>在超速离心机中，在4℃下以100,000xg离心20分钟。 </li><li> 离心后，从每个管中取出3/4的上清（ 例如 ，起始体积为60μL的45μL），并与1/3体积的4x样品缓冲液（在这种情况下为15μL）混合在一个单独的微量离心管中。 <ol><li>用凝胶加载尖端去除剩余的上清液，注意不要打扰颗粒（可能是玻璃状斑点）。 注意:在离心机运行完成后尽快从管中除去上清液以限制分离后的蛋白质解离是重要的。同样的原因，不要用反应缓冲液清洗沉淀物。 </li></ol></li><li> 向每个颗粒添加4/3体积的1x样品缓冲液t（如果起始体积为60μL，则为80μL）。 注意:这使得稀释与上清液相同（步骤4.8，加入1/3体积的4x样品缓冲液），允许沉淀和上清液样品之间的直接比较和测定造粒的蛋白质的百分比。 <ol><li>将样品缓冲液加入所有试管中，并在室温孵育至少5分钟。让颗粒置于样品缓冲液中以改善样品回收率。 </li><li>用p200移液管尖端将样品研磨8-10次，通过连续洗涤管的颗粒区域重新悬浮沉淀。在研磨过程中，将移液管末端轻轻刮去，以帮助重悬。 注意:注意避免在研磨过程中将空气引入样品，因为这将导致样品缓冲液起泡，并会降低样品回收率。 </li><li>研磨后将再悬浮的样品转移到微量离心管中。 </li> &lt;/醇&gt; </li><li>通过SDS-PAGE和考马斯染色8分析上清液和沉淀样品，每泳道加载10-15μL样品;这足以使蛋白质可视化。 注意:与F-肌动蛋白共沉淀的蛋白质将在"无F-肌动蛋白"颗粒样品中富集"加F-肌动蛋白"颗粒样品（ 图1A ）。如果蛋白质浓度在0.1-10μM范围内，则标准考马斯蓝染色足以检测。如果使用较低的蛋白质浓度来测量更高亲和力的相互作用，则可以使用胶体考马斯9或Western印迹来提高灵敏度。 </li><li>图像使用扫描仪或成像系统的考马斯染色凝胶（步骤5.12）。 </li></ol>造粒测定 - 定量注意:如果观察到与F-肌动蛋白的特异性结合，则可以测量t的亲和力他互动。这是通过对第4节中概述的协议进行一些修改和补充来实现的。有关设计和解释结合测定的优秀指南，请参阅Pollard 10 。提供了流程图（ 图2 ），以帮助分析和量化。 <ol><li>确定要测试的浓度范围。 注意:浓度范围将取决于蛋白质，并且应该从低于表观K d （ 例如 1μM）的浓度延伸到足够高的饱和结合浓度。重要的是，在浓度范围的高端，多个样品的结合达到饱和，以产生精确的结合曲线（ 图1C ）。如上所述，许多肌动蛋白结合蛋白在低微摩尔范围（1-5μM）中对F-肌动蛋白具有亲和力。对于K d为0.5-1μM的蛋白质，有用的起始浓度范围为0.1-10μM。 </li><li>硬旋转（步骤2.3）感兴趣的蛋白质去除聚集体。连续稀释蛋白质，使含有7-8个样品的浓度系列以2x的最终浓度测试。例如，如果要测试的范围为0.1-8μM，则在1x反应缓冲液中制备以下稀释液:16,8,4,2,1,0.5和0.2μM。 注意:如步骤4.2中所述，如果添加的蛋白质占第一稀释度的10％-20％（上述实施例中的16μM样品），可能需要进一步浓缩蛋白质或透析蛋白质转变成1x反应缓冲液。确保为"加F-肌动蛋白"和"无F-肌动蛋白"样品准备足够的每种稀释液。 </li><li> 通过在超离心管中的1x反应缓冲液中稀释目的蛋白质至所需浓度来制备样品（如第4部分）。保持样品体积低（40-60μL），以避免使用大量的蛋白质通过使用超薄具有最小体积（ 例如 ，每个保持0.2mL的7×20mm管）的步枪管。 <ol><li>将F-肌动蛋白添加到合适样品的所需最终浓度，并使用1x反应缓冲液调出体积。例如，对于使用2μMF-肌动蛋白的50μL反应，确保每个样品具有25μL的2x蛋白质，10μL的10μMF-肌动蛋白和15μL的1x反应缓冲液。包括对照样品（步骤4.4.2和4.4.3）。 注意:对于阴性和阳性对照，使用要测试的范围内的一个浓度（步骤5.1），理想情况下，接近中间至高端范围（如果浓度范围为0.1-10μM，则为4μM）。 </li></ol></li><li>在室温下孵育所有样品30分钟。 </li><li> 30分钟后，取出每个样品的1/5（ 例如， 10μL的50μL反应），并与20μL的水和10μL的4x样品缓冲液混合。 注意:这些是"总计"将用于生成标准曲线。 </li><li>将样品装入超速离心机转子。在100,000 xg和4℃下离心20分钟。 </li><li>离心后，取出3/4的上清液（ 如离心体积为40μL，每孔30μL），并与4x样品缓冲液（在这种情况下为10μL）混合，并分离出离心管。用凝胶加载尖端去除剩余的上清液，注意不要打扰沉淀。 注意:当测量结合亲和力时，不需要运行上清液。然而，保存上清液可能是有用的，特别是在测试新蛋白质时。 </li><li>如果不分析，则取出上清液（步骤5.7）。 </li><li> 将沉淀重悬于1体积的1x样品缓冲液中（如果离心体积为40μL，则为40μL）。 <ol><li>将样品缓冲液加入所有试管中，并在室温孵育至少5分钟。 </li><li> TR用p200吸头提取样品8-10次，连续洗涤管的颗粒区域。在研磨过程中，将移液管末端轻轻刮去，以帮助重悬。 </li></ol></li><li>将再悬浮的蛋白质转移到微量离心管中。 注意:这些是"Pellet"样品。 </li><li>通过SDS-PAGE 8分析Total和Pellet样品。如果可能，运行一个凝胶上的所有样品;如果没有，在一个凝胶上运行颗粒样品，并在一秒钟内运行总样品。 注意:鉴于样品数量，推荐使用大型凝胶系统进行分析。如果在两个或多个凝胶上运行样品，重要的是所有凝胶都被染色相同（ 即，相同的考马斯溶液和相同的染色/脱色时间）。 </li><li>图像考马斯染色的凝胶使用成像系统，测量宽（ 即，两到三对数）和线性范围内的蛋白质带强度。确保图像a没有饱和像素收集。 注意:基于激光的成像系统提供最佳的灵敏度和信噪比。 </li><li> 使用ImageJ或类似的分析程序，测量蛋白质带强度并计算结合蛋白的量。 注意:对于所有样本测量，使用ImageJ中的选择工具绘制每个频带周围的感兴趣区域（ROI），并测量（分析&gt;测量）区域和平均灰度值。通过从没有样品的区域测量平均灰度值来计算每个凝胶的背景。从每个ROI平均灰度值减去背景平均灰度值，然后乘以面积以获得每个频带的积分密度值。 <ol><li>从总样本测量感兴趣的蛋白质带强度（ 图2A ）。 </li><li>通过绘制带强度（ 即综合密度测量）与蛋白质质量的关系来生成标准曲线（ 图2B）。 </li><li>测量与F-肌动蛋白共沉淀的感兴趣蛋白质的量（共沉淀蛋白， 图2C ）。 </li><li>测量在不存在F-肌动蛋白时沉淀的感兴趣的蛋白质的量（背景沉淀， 图2D ）。 </li><li>从共沉淀蛋白中减去背景沉淀（ 即从步骤5.13.3中减去步骤5.13.4的值），以确定与F-肌动蛋白结合的蛋白质的量。 </li><li>测量每个颗粒中的F-肌动蛋白的量（ 图2E ）。确定每个样品的平均F-肌动蛋白量，然后将每个样品除以平均值，以确定每个样品中F-肌动蛋白与平均值之比（ 即，低于条带的数字）。 </li><li>对于每个样品，通过F-肌动蛋白肌动蛋白颗粒比（步骤5.13.6）将结合的蛋白质的量（在步骤5.13.5中计算）分配以调整颗粒中的差异。 ñOTE:该值是归一化的结合蛋白。 </li><li>使用标准曲线（步骤5.13.2）计算每个样品中标准化结合蛋白的量（质量）（步骤5.13.7）。 注意:最初去除的蛋白质（"总"样品）以及负载量（除了整个颗粒负载以外，将是颗粒的一部分），必须在计算蛋白质总量时考虑那颗颗粒。 </li><li>从颗粒中的蛋白质总量（步骤5.13.8中计算）和样品的体积确定每个样品中结合蛋白的浓度。从起始浓度减去该值以确定游离蛋白的量。通过肌动蛋白的浓度（肌动蛋白的μM/μM）和阴性相对于游离蛋白的浓度分离结合蛋白的浓度以产生结合曲线（ 图1C ）。 注意:由于F-肌动蛋白不是单一的，均匀的物种，它是diffi从G-肌动蛋白浓度推断F-肌动蛋白的摩尔浓度。使用起始G-肌动蛋白浓度来确定结合蛋白的量（肌动蛋白的μM/μM），并估计反应中结合位点的表观浓度。结合位点的浓度通常小于反应中肌动蛋白单体的浓度，因为不是所有的肌动蛋白聚合，并且因为单个肌动蛋白结合蛋白分子可以与肌动蛋白丝上的多个单体接触。 </li></ol></li><li>使用统计程序，通过使用非线性最小二乘回归从结合曲线确定亲和力（K d ）和B max 。 注意:Scatchard图不鼓励用于分析结合数据，部分原因是它们可能会掩盖绑定是否饱和，并且可能会扭曲实验错误10 。 </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+actin:+purification+and+single+molecule+assembly+assays.">Cytoplasmic actin: purification and single molecule assembly assays.</a> Methods Mol Biol. 1046, 145-170 (2013).</li></ol>

作者宣称他们没有竞争的经济利益。

肌动蛋白共沉淀测定是一种直接的技术，可以快速确定蛋白质是否结合F-肌动蛋白。通过一些修改，该技术也可以用于测量交互的亲和力。除了上述方案中提出的观点外，在设计，实施和解释测定时应考虑以下问题。 感兴趣的蛋白质新鲜制备的或冷冻的蛋白质可用于测定。如果使用冷冻蛋白，强烈建议将结果与新鲜（未冷冻）蛋白进行比较，以确保冷冻不影响F-肌动蛋白结合。 G-肌动蛋白来源许多造粒实验使用从肌肉中分离的G-肌动蛋白，因为其相对丰度。在哺乳动物中有三种主要的肌动蛋白同种型 - α，β和γ - 是非常相似的（&gt; 90％序列识别TY）。尽管如此，在同种异型12,13之间存在功能差异。如果可能，结合测定中使用的G-肌动蛋白同种型应与体内同种型相符。例如，如果测试在骨骼肌中表达的蛋白质，则α-肌动蛋白是最佳选择;如果检查在成纤维细胞中表达的蛋白质，推荐使用β-肌动蛋白。 鬼伞素使用由于鬼笔环肽结合F-肌动蛋白，它可能会干扰甚至阻断一些F-肌动蛋白结合蛋白（ 例如，鬼笔环肽嵌段cofilin与肌动蛋白丝结合）的结合。因此，谨慎使用鬼笔环肽，如果可能，结果与非鬼笔环己烷处理的样品相比。 高背景蛋白质在不存在F-肌动蛋白的情况下沉淀是常见的（ 图1A ，没有F-肌动蛋白颗粒样品S）。然而，高水平的背景沉淀可以掩盖真正的肌动蛋白共沉淀，并且使得难以（如果不是不可能）确定蛋白质是否结合F-肌动蛋白或测量相互作用的亲和力。向反应缓冲液中加入polidicanol（步骤4.1）可以显着降低背景，是一个简单的解决方案。如果不降低背景，调整反应缓冲液，盐浓度和/或培养温度可能有所帮助。 绑定曲线为了产生结合曲线，有必要在一系列反应中改变感兴趣的蛋白质或F-肌动蛋白的浓度。在实践中，将F-肌动蛋白维持在固定浓度并且改变目的蛋白质的浓度是更容易和优选的。在造粒测定中以固定浓度（ 例如 2μM）维持F-肌动蛋白限制在较高浓度的F-肌动蛋白上的非特异性捕获，并且防止在低于（&lt;0.5μM）的F-肌动蛋白浓度下解聚。可以使用鬼笔环肽来防止解聚，尽管这引入了系统中潜在的复杂因素（见第3.3及以上）。维持固定浓度的F-肌动蛋白还可以使样品间的F-肌动蛋白颗粒进行比较（并标准化），并鉴定失败的实验（ 即 F-肌动蛋白颗粒高度可变，防止浓度分析）。最后，将F-肌动蛋白维持在固定的浓度允许确定与肌动蛋白丝的结合是否合作（ 图1C ）。 饱和结合如在所有结合实验中，与F-肌动蛋白的结合是饱和的以及蛋白质加上F-肌动蛋白平台的浓度是至关重要的（ 图1C ）。没有平台，不可能计算准确的解离平衡常数。因此，它仔细规划待测试的稀释系列并且始终包含较高浓度的蛋白质（ 即，比预期的K d高至少5至10倍）是重要的。 绑定分析为了使测量的解离常数是确定性的，应该使用允许将F-肌动蛋白的结合位点浓集在感兴趣的蛋白质上的F-肌动蛋白浓度远低于亲和力来进行测定。为了检查是否符合此标准，估算B max的结合位点浓度。例如，如果[F-肌动蛋白]为2μM，B max = 0.5，则[结合位点]≈1μM。 K d应至少比[结合位点]大5至10倍。如果测量的K d与[结合位点]具有相同的数量级，则观察到的结合曲线可能表示高亲和力结合si的滴定而不是真正的结合等温线。如果观察到这一点，请使用10倍低F-肌动蛋白浓度重复测定以测量准确的亲和力。对于高亲和力相互作用，为了实现足够低的F-肌动蛋白浓度以精确测量亲和力，可能需要鬼笔环肽稳定（步骤3.3）。 最后，研究人员在进行和评估测定时应注意共沉淀测定的基本限制。最重要的是，共沉淀测定不会产生真正的平衡常数。在离心过程中将结合产物（ 即蛋白加F-肌动蛋白）与反应物分离，随后产物可以解离以产生新的平衡。结果，共沉淀测定可能会错误计算或不能检测到低亲和力相互作用。由于许多肌动蛋白结合蛋白对F-肌动蛋白具有低（ 即微摩尔）亲和力，所以阴性结果<em&gt;即，无可检测的结合）不一定意味着蛋白质不结合F-肌动蛋白。作为替代方案，基于TIRF显微镜的单丝结合测定对于确定解离常数更灵敏和更准确（对于该技术的评论，参见参考文献15,16 ）。尽管有这些限制，造粒测定是大多数研究人员的手段，并且是确定蛋白质是否结合F-肌动蛋白并测量相互作用的亲和力的有效工具。

肌动蛋白是必需的细胞骨架蛋白，在多种细胞过程中起关键作用，包括运动，收缩，粘附和形态1 。肌动蛋白以两种形式存在:单体球状肌动蛋白（G-actin）和聚合丝状肌动蛋白（F-肌动蛋白）。在细胞内，F-肌动蛋白组织由大量蛋白质控制，其调节肌动蛋白丝2,3,4的成核，生长，交联和分解。然而，多种肌动蛋白结合蛋白如何协调一致地调节肌动蛋白网络组织仍然在很大程度上不清楚。 蛋白质 - 蛋白质相互作用的测量是了解蛋白质在生物化学水平上如何发挥其对细胞行为的影响的重要途径。许多不同的测定法可用于检测纯化蛋白质之间的相互作用。可溶性蛋白质的常见方法包括下拉，荧光偏振，等温滴定量热法和表面等离子体共振。重要的是，所有这些测定都需要蛋白质可溶，因此难以适应与聚合物丝状蛋白质如F-肌动蛋白一起使用。在这里，我们描述了一种技术 - 肌动蛋白共沉淀或造粒，测定 - 以确定蛋白质或蛋白质结构域是否结合F-肌动蛋白并测量相互作用的亲和力。 肌动蛋白造粒测定是一种相对简单的技术，除了超速离心机外，不需要专门的设备。所有的试剂都可以根据基础生物化学知识或购买。一旦与F-肌动蛋白结合，该测定可用于测量表观亲和力（ 即解离平衡常数）。此外，一旦建立亲和力，就进行造粒测定是测量感兴趣蛋白质（ 即翻译后修饰，突变或配体结合）的变化如何影响其与F-肌动蛋白6的相互作用的有用工具。该技术在尝试测定之前确实存在研究人员应该注意的限制（参见讨论 ）。

<table><tbody><tr><td>Sorvall MTX 150 Micro-Ultracentrifuge</td><td>ThermoFisher Scientific</td><td>46960</td><td></td></tr><tr><td>S100-AT3 rotor</td><td>ThermoFisher Scientific</td><td>45585</td><td></td></tr><tr><td>Ultracentrifuge tubes - 0.2 mL</td><td>ThermoFisher Scientific</td><td>45233</td><td></td></tr><tr><td>Actin, rabbit skeletal muscle</td><td>Cytoskeleton</td><td>AKL99</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A8531</td><td></td></tr><tr><td>Polidicanol (Thesit)&amp;nbsp;</td><td>Sigma</td><td>88315</td><td></td></tr><tr><td>Phalloidin</td><td>ThermoFisher Scientific</td><td>P3457</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>ThermoFisher Scientific</td><td>R0862</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>Imidazole</td><td>Fisher Scientific</td><td>O3196</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>M33</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma</td><td>3779</td><td></td></tr><tr><td>Odyssey CLx Imaging System</td><td>LI-COR</td><td></td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250 Dye</td><td>ThermoFisher Scientific</td><td>20278</td><td></td></tr><tr><td>Colloidal Blue Staining Kit</td><td>ThermoFisher Scientific</td><td>LC6025</td><td></td></tr></tbody></table>

measuring protein binding to f-actin by co-sedimentation

细胞内的丝状肌动蛋白（F-actin）组织受大量控制肌动蛋白成核，生长，交联和/或拆解的肌动蛋白结合蛋白的调节。该方案描述了一种技术 - 肌动蛋白共沉淀或造粒，测定 - 以确定蛋白质或蛋白质结构域是否结合F-肌动蛋白并测量相互作用的亲和力（ 即解离平衡常数）。在该技术中，首先将感兴趣的蛋白质与溶液中的F-肌动蛋白一起温育。然后，使用差速离心法沉淀肌动蛋白丝，通过SDS-PAGE分析沉淀物。如果感兴趣的蛋白质结合F-肌动蛋白，它将与肌动蛋白丝共沉淀。可以量化结合反应的产物（ 即， F-肌动蛋白和目的蛋白）以确定相互作用的亲和力。肌动蛋白造粒测定法是一种简单的确定方法如果感兴趣的蛋白质结合F-肌动蛋白，并评估该蛋白质如配体结合的变化如何影响其与F-肌动蛋白的相互作用。

我们检查了在共沉淀测定中αE-连环蛋白同源二聚体结合F-肌动蛋白。由于过去的实验已经表明，α-catenin同二聚体对F-肌动蛋白的亲和力约为1μM，B max在11 11附近，我们用低浓度的F-肌动蛋白（0.2μM而不是2μM）进行测定讨论）。由于0.2μM低于临界浓度，所以加入鬼笔环肽以稳定从兔骨骼肌G肌动蛋白聚合的F-肌动蛋白（步骤3.3）。在0.2μMF-肌动蛋白存在或不存在下培养增加浓度的αE-连环蛋白同二聚体（0.125-12.0μM）。将样品离心，并分析得到的颗粒（ 图1A ）。如预期的那样，αE-连环蛋白同二聚体与背景中的F-肌动蛋白共沉淀（ 图1A ，将F-肌动蛋白颗粒样品与无F-ac锡丸样品）。 BSA作为阴性对照（ 图1B ）。结合蛋白质被定量并绘制在游离蛋白上，以计算相互作用的亲和力（ 图1C ）。绘制的数据最适合Hill方程。计算的K d为2.9μM，B max为0.2，Hill系数（h）为4.8。因此，αE-连环蛋白同源二聚体以低微摩尔亲和力协同结合F-肌动蛋白，与以前的工作（2.9μM对〜1.0μM的K d ） 11一致 。 <img alt="图1" src="/files/ftp_upload/55613/55613fig1.jpg" /> 图1: 高速F-肌动蛋白共沉淀测定。 （ A ）将增加的浓度（0.125-12.0μM）的αE-连环蛋白同源二聚体与（左图）或没有（右图）一起孵育0.2μM用肌钙磷稳定的F-肌动蛋白ñ。将它们在室温下孵育30分钟并离心。通过SDS-PAGE分离总量（起始原料的7.5％）和沉淀物（50％的沉淀物），并用考马斯染料染色。 （ B ）作为阴性对照，运行4μMBSA。通过SDS-PAGE分离具有（+）或不含（ - ）F-肌动蛋白的总和颗粒样品，并用考马斯染料染色。 （ C ）从游离αE-连环蛋白（μM）绘制来自A的结合αE-连环蛋白（μM/μM肌动蛋白），数据拟合到Hill方程（红线）。列出了K d ，B max和Hill系数（h）。 <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55613/55613fig2.jpg" /> 图2: 肌动蛋白造粒定量 </strong&gt; - 流程图。该示意图概述了第5节中的关键步骤，其中包括用于定量的总计和颗粒样品（ A ， CE ）和标准曲线（ B ）的实例。 5.13步骤: 1 ）测量总样品（A）中感兴趣的蛋白质的量。 2 ）通过绘制带强度与蛋白质质量（B）的关系来生成标准曲线。 3 ）测量与F-肌动蛋白（ C ）共沉淀的感兴趣的蛋白质的量。 4 ）测量不存在F-肌动蛋白（ D ）时造粒的蛋白质的量。 5 ）从C中减去D以确定与F-肌动蛋白结合的蛋白质的量。 6 ）测量每个颗粒（E）中的F-肌动蛋白的量，计算每个样品的平均F-肌动蛋白量，并将每个样品除以平均值（下面的数字显示比例）。 7 ）对于每个样品，将结合的蛋白质的量（步骤5中计算）除以F-肌动蛋白颗粒比（在步骤6中计算）以调整颗粒中的差异。 8 ）使用标准曲线（ B ）计算每个样品中标准化结合蛋白的量（质量）（步骤7）。 9 ）确定游离蛋白和结合蛋白的浓度以产生结合曲线。 <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

Measuring Protein Binding to F-actin by Co-sedimentation

该方案描述了一种测试蛋白质与丝状肌动蛋白（F-肌动蛋白）共沉淀的能力的方法，并且如果观察到结合，则测量相互作用的亲和力。

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

measuring-protein-binding-to-f-actin-by-co-sedimentation

Research

JoVE Journal

Biochemistry

16.4K 次观看.  University of Pittsburgh School of Medicine. 该方案描述了一种测试蛋白质与丝状肌动蛋白（F-肌动蛋白）共沉淀的能力的方法，并且如果观察到结合，则测量相互作用的亲和力。 

视频: Measuring Protein Binding to F-actin by Co-sedimentation

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小型低聚物形成膜蛋白的沉降平衡:组氨酸质子对五聚体稳定性

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

化学

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

生物学

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

用市售蛋白质重建基于肌动蛋白的运动

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

生物工程

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ的聚合反应检测：简单的协议和注意事项

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

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

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

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

生物分子缩合物中蛋白质相互作用动力学的单分子测量

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

通过共沉淀测量蛋白与F-肌动蛋白的结合

摘要

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此视频中的章节

荧光各向异性作为一种工具来研究蛋白质相互作用

小型低聚物形成膜蛋白的沉降平衡:组氨酸质子对五聚体稳定性

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

通过荧光体积排阻色谱快速评估膜蛋白质量

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

用市售蛋白质重建基于肌动蛋白的运动

FtsZ的聚合反应检测：简单的协议和注意事项

生物分子缩合物中蛋白质相互作用动力学的单分子测量

通过共沉淀测量蛋白与F-肌动蛋白的结合

摘要

探索更多视频

此视频中的章节

荧光各向异性作为一种工具来研究蛋白质相互作用

小型低聚物形成膜蛋白的沉降平衡:组氨酸质子对五聚体稳定性

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

通过荧光体积排阻色谱快速评估膜蛋白质量

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

用市售蛋白质重建基于肌动蛋白的运动

FtsZ的聚合反应检测：简单的协议和注意事项

生物分子缩合物中蛋白质相互作用动力学的单分子测量

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析