这项工作是由教育部通过国家需求计划（P200A100044）领域的研究生援助支持。

注意:此协议需要放射性物质在使用适当的培训和处理。执行此法时，包括β辐射的屏蔽，请使用必要的个人防护装备。放射性废物必须小心处理，因为在实验过程中的清洗阶段会产生大量的浪费。确保废物贮存在一个直立的容器，如大型桶或瓶子，不会轻易被撞倒或溢出。一旦实验结束后，小心地转移所有的液体废物适当标识放射性废物容器。处理所有材料小心，并保持一个盖格计数器附近的监控污染的工作空间。 注:此协议是我们组先前报道的实验的修订版。在H 3 PO 4 15 Phosphohistidine稳定性应该为之前使用此方法的任何未表征组氨酸激酶进行试验。该phosphohistidine ST能力测试已如前所述。 15不包含激酶阴性对照必须包括在内。这是必要的，以便减去来自每个样品的背景信号，并确保[γ-32 p] -ATP充分从膜洗涤。 1.试剂和材料的制备<ol><li>从细菌培养使用这种检测前净化组氨酸激酶。 <ol><li>净化从副溶血性弧菌 （EB101，ATCC 17802）组氨酸激酶（基因ID 1189383）用于该测定法的发展。克隆用NdeI和XhoI限制性位点激酶入表达载体pET-23aHis-TEV，并进行定点诱变以产生突变体构建Vp1876 D499A。 </li><li>变换质粒入大肠杆菌 BL21（DE3）pLysS中，并在37℃下生长的细胞中TB介质。补充培养物用氨苄青霉素（100微克/毫升）和氯霉素（34微克/毫升），并在搅拌下生长（250rpm）以的OD 0.6 600。 </li><li>诱导用IPTG蛋白表达于25μm的终浓度，并在16℃下生长培养物过夜。收获细胞离心（5000 XG），通过超声裂解和离心机除去细胞碎片（18,000 XG）。 </li><li>纯化使用Ni-NTA琼脂糖的His-标记的激酶。确认通过SDS-PAGE的纯度。优化纯化对于每种蛋白质，并确认之前使用该测定纯度。 </li></ol></li><li>准备25毫米H 3 PO 4洗涤液。到1升的去离子蒸馏水中，添加H 3 PO 4至25毫米的最终浓度。此溶液的pH值是大约2.0。将25毫米H 3 PO 4冰上。 </li><li>制备的325毫米的 H 3一13倍原液PO 4，以用来骤冷激酶反应。该溶液的pH为约1.5。将325毫米H 3 PO 4冰上。 注:H 3 PO 4 </sub&gt;在25mM的最终浓度足以淬灭反应，并还有助于阻止不从结合到硝酸纤维素膜结合到组氨酸放射性标记的磷酸盐。 </li><li>制备含160毫摩尔Tris -盐酸pH值8.0，600mM的氯化钾，16毫MgCl 2的4倍反应缓冲原液，和40％甘油。 </li><li>量化设立的蛋白浓度的方法（布拉德福德，紫外-可见光等 ），组氨酸激酶的浓度。 16,17准备用浓度是所需的终浓度的4倍一个组氨酸激酶溶液。以获得足够的信号，在反应的最终蛋白质浓度最好应至少为5微米。 </li><li>制备了4倍的放射性标记的[γ-32 P]与未标记的ATP的适当4倍浓度和标记-ATP溶液:未标记的比率为实验。 注:应包含在该混合物[γ-32 p] -ATP的量依赖于多少[^7; - 32 P] -ATP最终会发现每个反应。我们已获得足够的信号与终浓度为0.1之间 - 每个反应微居里5。重要的是，该标记:未标记的ATP比来在给定的实验中的所有反应保持恒定。当多个ATP浓度所需的连续稀释应，因为这也保持标记:在所有未标记的浓度ATP比值不变。最终的ATP浓度范围通常从10μM到至少1毫米，并且每个浓度应一式三份，以获得可靠的动力学数据来测定。 </li><li> 96孔点印迹装置<ol><li>切8厘米×12厘米片硝酸纤维素膜（孔径0.2μm）的。 </li><li>位置硝化纤维素在斑点印迹装置中，确保该膜适合，使得该装置是密封的，并且所有孔中由膜完全覆盖。如果组装正确，不应该有空气泄漏时的真空是申请灭蝇灯。 </li><li>收集滤液，该装置连接到一个次级侧臂烧瓶中。从阀杆，连接适当管道，使得该装置可以在不翻倒辅助烧瓶舒适地使用。 </li><li>连接次级侧臂烧瓶中，吸气器/真空源。 </li><li>测试该装置完全被施加真空到该装置密封。如果该装置被正确地组装时，一个强的真空将存在于所有孔中。所有的管道连接，可以包裹在封口膜或保鲜膜，保证密封。 </li><li>任选地，测试真空，吸管100微升反应缓冲液为一良好。真空应当足够强通过井和到膜上绘制所有液体。 </li><li>关闭真空，直到所有样品准备在膜上要看准。 </li></ol></li></ol> 2.反应开始淬火<ol><li>反应组分的混合<ol><li>此前initi通货膨胀，准备所有四个反应成分为4倍的股票解决方案:反应缓冲液（参见1.4节），组氨酸激酶（1.5），[γ-32 P] -ATP（1.6），和双蒸2 O. </li><li>混合反应缓冲液，组氨酸激酶等体积及ddh 2 O允许这些反应组分在室温下平衡的时间很短（10分钟）。 注:最终反应体积是依赖于实验是否是时间依赖的，依赖酶，或衬底依赖性。时间相关的反应必须是较大，因为多个等分试样从相同反应采取并淬火在期望的时间点。反应体积将取决于所希望的时间点的数量。该试验为在硝酸纤维素膜上各点以包含在反应30微升优化。因此，如果10个时间点都需要，反应体积必须至少300微升（较高的音量如330微升将在任何移液误差的情况下，优选的）。酶和底物依赖性的实验需要较小的反应体积。这些实验的反应体积可为30微升的小，因为这是将在硝酸纤维素膜上点样的最终体积。 </li><li>以引发反应，将[γ-32 P] -ATP溶液到反应中的一个体积并通过上下抽吸混合。曲目经过反应时间用计时器，并允许反应进行所需的时间。 </li><li>淬灭反应，加入1/13冰冷325毫H 3的总反应体积PO 4的反应，并通过上下抽吸混合。备选地，添加反应至325毫H 3 PO 4的等分试样。 注:的H 3 PO 4的最终浓度应为25mM至足以淬灭反应。例如，添加2.5微升325毫米H 3 PO 4〜30微升的反应，或加30μL反应，2.5微升325毫米H 3 PO &lt;子&gt; 4。反应的最终pH为约4.0。的H 3 PO 4这个浓度已经过测试，证实淬火该缓冲区的激酶反应，而不会降低phosphohistidine。 <ol><li>例如，混合7.5微升4倍的反应缓冲液，7.5微升4倍的组氨酸激酶，和7.5微升的DDH 2 O.启动与7.5微升[γ-32 P] -ATP反应。淬灭，用2.5微升325毫H 3 PO 4的反应。 </li></ol></li><li>立即将淬火反应冰上直到所有的反应已经终止。 注意:为了最大限度地提高效率，最好以引发一个反应每隔15或20秒，直到所有的反应都启动，并且一旦所需反应时间已过，淬火一种反应每隔15或20秒，直到所有被骤冷。对于那些谁正在使用的测定法的第一次，较长的间隔可以更易于管理。 </li></ol></li></ol> 3.去渍Ø硝酸纤维素˚F淬火反应<ol><li>在96孔点印迹装置启动真空<ol><li>放置预组装96孔斑点印迹装置（部分1.7）转换成次级容器。此容器应足够大的装置可以很容易地拆卸中，作为装置和膜将使用后的放射性。 </li><li>施加真空到96孔斑点印迹装置。 </li><li>一旦该装置是在真空下，小心地直接吸管猝灭反应到在每个硝酸纤维素膜良好。重复，直到所有的反应都装载到膜上。由于硝化纤维素的结合力（75 - 110微克/厘米2）是比点样，它可以假设正确装载时，几乎所有的激酶结合到膜上的组氨酸激酶的量。 注意:根据所使用的装置中，必须小心不要刺破硝化纤维素。观察所有的反应已经与日接触Ë硝化棉。很容易让一些或所有的反应的粘到孔的壁上，并且不会到达硝化纤维素。 </li><li>用100微升冰冷的25 mM的H 3 PO 4的洗井。这将允许可能已截留在井到达膜的任何反应体积，以确保所有的反应的完全加载。允许整个卷到通过膜流动。 </li></ol></li><li>设备拆卸和硝酸纤维素膜转移<ol><li> 小心拆卸切断真空之前的装置。注意，这两个装置与硝化纤维素膜是在这个时候放射性。从二级容器在这个时候不要删除任何设备组件。 </li><li>用钳子，小心地将硝酸纤维素膜从该装置转移到约200mL的25mM的冰冷的 H 3 PO 4的容器。把这个容器上的盖子，因为洗必拉迪oactive。此时，真空可以被关闭。 </li></ol></li></ol> 4.硝化棉处理<ol><li>洗硝化棉<ol><li>将容器与摇杆洗涤膜。允许该膜轻轻20分钟摇动。 </li><li> 20分钟后， 小心地倒出所用洗涤溶液放入大水桶用于临时废弃物存储。加入200毫升冰冷的25mM H 3 PO 4的向膜并重复。 注:至少有三个20分钟的洗涤是必要的，以去除背景[γ-32 P]从膜-ATP信号。第三次洗涤后，测试用盖革计数器辐射所使用的洗涤液。继续以上直到没有信号存在于洗涤溶液中所描述的洗膜。 </li></ol></li><li>干燥硝化棉<ol><li>将膜充分洗涤后，使膜简要空气干燥。这个过程通常需要5分钟以下。</li></ol></li></ol> 5.暴露于存储荧光屏注意:此部分是可选的。该膜暴露于荧光屏是因为它允许对放射性标记的激酶在对膜的每个斑点的强度的可视化是有益的。这些斑点的相对强度成正比磷酸组氨酸激酶，在每个点的量。强度可与图像处理软件进行量化，并且这些结果可以进行比较，以从部分7另外产生的那些，该步骤允许进行质量控制。在此扫描可见异常或许可以解释从闪烁计数获得非常规的结果。 <ol><li>荧光屏准备<ol><li>前的膜暴露到存储荧光屏，露出荧光屏白色光为至少5分钟。这确保了可以由屏幕举行任何残留图象被擦除。 </li><li>确保屏幕干净和干燥。如果有必要，轻轻地清洁与屏幕制造商批准的荧光屏清洗液的屏幕，并擦干。 </li></ol></li><li>硝酸纤维素膜的制备<ol><li>在一个薄的塑料膜小心包住干硝酸纤维素膜，以防止损坏荧光屏残留水分。确保没有褶皱或气泡。 </li></ol></li><li>暴露于荧光屏<ol><li>放置包裹的硝酸纤维素膜在存储荧光屏盒，放置在屏幕正面朝下到膜上，并关闭磁带。不要打开纸盒或直到它是时间来扫描荧光屏，如同置身在膜转移会造成双重或模糊图像被捕获移动膜。 </li><li>暴露至少4小时，或只要在荧光屏制造商建议。 </li><li>一旦完成曝光，扫描荧光体屏幕并有荧光扫描器捕获图像。 </li></ol></li></ol> 6.准备膜硝化棉闪烁计数<ol><li> Ponceau S染色和脱色<ol><li>简要地沉浸在Ponceau S染色溶液中的硝酸纤维素膜（0.1％丽春红S（重量/体积）在5％乙酸）为1 - 2分钟。湿之前脱色污渍整个膜。 </li><li>从膜倒出过量丽春红硫。 </li><li>冲洗DDH 2 O的膜，直到仅从组氨酸激酶的斑点染色。需要注意的是，如果所有斑点含有蛋白质的检测量的此过程才有效。 注意:此步骤是必要的，以确认该激酶结合到硝酸纤维素，并且该蛋白质加载甚至在所有斑点。它也允许斑激酶从膜容易地切出。 </li></ol></li><li>切割出点<ol><li>使用剪刀，切出在硝化纤维素膜上各点。它没有必要完全切断ALON克染色斑的边缘;如果膜充分洗涤，背景由于切出斑点的大小不同将是可忽略不计。切出每个反应的整个点是唯一重要的。 </li><li>使用镊子，每个点仔细转移到闪烁瓶。无闪烁鸡尾酒是必要的，因为32 P为切伦科夫辐射易被察觉。 注意:可替换地，光点可以用一个尖锐的打孔器切出。从与打孔器硝化棉删除每个点，并将其推入一个销闪烁瓶中。用这种方法，在膜不需要触及，进一步减少的辐射暴露。 </li></ol></li><li> [γ-32 P] -ATP解决方案的确定CPM /皮摩尔<ol><li>现货几个稀释[γ-32 P] -ATP溶液（1.5节）到1厘米硝化棉×1cm的正方形。简单地说风干。 </li><li>使用镊子，每平方米仔细转移到闪烁小瓶。没有闪烁鸡尾酒是必要的。这些样本将被用来产生标准曲线来确定ATP溶液的CPM /皮摩尔。 </li></ol></li></ol> 7.闪烁计数<ol><li>加载所有闪烁瓶到闪烁计数器录像带。装载盒引入闪烁计数器。 </li><li>执行闪烁计数方案，测量每个小瓶CPM。这些数据，与放射性标记的ATP混合物（从节6.3）的CPM /皮摩尔沿，可以用来计算磷酸组氨酸激酶的存在于每一个点的数量，并因此自磷酸化的速率。 18,19 </li></ol>

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R., 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=Monoclonal+1-+and+3-Phosphohistidine+Antibodies:+New+Tools+to+Study+Histidine+Phosphorylation.">Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.</a> Cell. 162 (1), 198-210 (2015).</li><li>Ueno, T. B., Johnson, R. A., Boon, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+assay+for+the+quantification+of+histidine+kinase+autophosphorylation.">Optimized assay for the quantification of histidine kinase autophosphorylation.</a> Biochem. Biophys. Res. Commun. 465 (3), 331-337 (2015).</li><li>Bradford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 7 (72), 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Funt, B. L., Hetherington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scintillation+counting+of+beta+activity+on+filter+paper.">Scintillation counting of beta activity on filter paper.</a> Science. 131 (3413), 1608-1609 (1960).</li><li>Bem, E. M., Bem, H., Reimch&#252;ssel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+phosphorus-32+and+calcium-45+in+biological+samples+by+&#268;erenkov+and+liquid+scintillation+counting.">Determination of phosphorus-32 and calcium-45 in biological samples by &#268;erenkov and liquid scintillation counting.</a> Int. J. Appl. Radiat. Isot. 31 (6), 371-374 (1980).</li></ol>

作者什么都没有透露。

我们所描述的硝化纤维结合分析具有比以前使用的方法很多优势表征组氨酸激酶。相较于传统的SDS /基于页面的放射自显影，我们的方法是更高的吞吐量和更少耗费时间。硝酸纤维素膜更容易处理比SDS凝胶，并且不需要是固定的。丽春染色硝化棉允许而被可视化的蛋白斑点。这提供了一种简单的方法来切出每个点的闪烁计数，并确定蛋白质加载在所有点是一致的。每个点的闪烁计数提供了可以使?...

适应性反应是细菌生存的关键。为了检测和环境的变化作出反应，细菌使用一种称为双组分信令的刺激反应系统。 1,2在典型的双组分体系中，组氨酸激酶检测同源的刺激，autophosphorylates其保守组氨酸残基，然后传送磷酸盐上的反应调节蛋白的接收机结构域的保守天冬氨酸残基。 3该事件触发的响应调节，刺激下游效应的活性的改变。 4,5-因此，细菌能够感知和适应本地环境的变化。一些双组分信号系统从这个原型偏离。在一些情况下，组氨酸激酶的感官域是一个独立的蛋白，它直接检测感知输入，并通过蛋白质 - 蛋白质相互作用修改激酶活性。 6 - 8然而，基金amental过程和系统的整体作用是相同的。双组分信令是一个普遍存在的刺激反应系统，该系统对细菌存活是必不可少的，和组氨酸激酶在信号转导中起关键作用。 9 尽管组氨酸激酶细菌生物学的重要性，他们仍然很少描述。这是由于phosphohistidine固有的不稳定性，以及缺乏用于测量自磷酸化的实用的方法。 Phosphohistidine比磷酸丝氨酸，磷酸苏和磷酸更不稳定。 10因此，通常用于分析丝氨酸/苏氨酸/酪氨酸激酶的技术是不适用的组氨酸激酶。 11 在体外测定来研究组氨酸激酶基本上都被限制在SDS-PAGE上放射自显影。 12,13在该方法中，[γ-32 p] -ATP一起温育与激酶和激酶的磷酸化是由聚合酶分析yacrylamide凝胶电泳（PAGE），随后该凝胶的放射自显影。此方法可用于监测激酶自身磷酸化，以及从所述激酶反应调节磷酸转移。然而，这种方法具有显着的缺点。基于页面的测定是低吞吐量和费时。这样的限制是不利于表征蛋白并确定其动力学参数。这是最近公布的研究组氨酸激酶的另一种方法是利用phosphohistidine抗体来检测磷酸化。 14虽然这种方法具有1- phosphohistidine和3- phosphohistidine区分的优点，这取决于用于检测的仪器，这种方法可能不提供大的动态范围或检测的高上限。因此，需要一种可用于研究这些重要的蛋白质一更快，更费力，更敏感的检测。 这里，我们描述以及demonstrate一个精心开发硝化纤维结合测定法可用于定量在体外纯化细菌组氨酸激酶的磷酸化。该测定是更高的吞吐量和耗时比基于PAGE的测定以下。该方法也利用了phosphohistidine量化，它提供了检测和大的动态范围的高上限切伦科夫辐射。该测定可用于确定用于组氨酸激酶动力学参数。

<table><tbody><tr><td>Phosphoric acid</td><td>VWR</td><td>AAAA18067-AP</td><td>For quenching reactions and washing nitrocellulose</td></tr><tr><td>Tris base</td><td>RPI</td><td>T60040-5000.0</td><td>Tris-HCl pH 8.0, for kinase reaction buffer</td></tr><tr><td>Potassium chloride</td><td>RPI</td><td>P41000-2500.0</td><td>For kinase reaction buffer</td></tr><tr><td>Magnesium chloride</td><td>RPI</td><td>M24000-500.0</td><td>For kinase reaction buffer</td></tr><tr><td>Glycerol</td><td>RPI</td><td>G22020-4000.0</td><td>For kinase reaction buffer</td></tr><tr><td>5&#039;-ATP</td><td>Promega</td><td>E6011</td><td>Kinase substrate</td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]-5&#039;-ATP</td><td>Perkin Elmer</td><td>NEG002Z250UC&amp;nbsp;</td><td>6,000 Ci/mmol</td></tr><tr><td>96-well dot blot apparatus</td><td>Bio-rad</td><td>1706545</td><td>For spotting reactions</td></tr><tr><td>Nitrocellulose</td><td>Whatman</td><td>32-10401396-PK</td><td>For spotting reactions</td></tr><tr><td>Ponceau S</td><td>Sigma aldrich</td><td>P3504-50G</td><td>For staining nitrocellulose</td></tr></tbody></table>

quantification of bacterial histidine kinase autophosphorylation using a nitrocellulose binding assay

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

<html>生成的代表性数据集配有荧光成像仪（ 图1）的硝化纤维素膜的，丽春红染色（ 图2），和闪烁计数数据（ 图3）拍摄的图像。 图3A示出了在双倒数图酶动力学常数。使用来自革兰氏阴性物种副溶血性弧菌 （基因ID 1189383）精制组氨酸激酶，获得这些结果。用于净化这种激酶的协议第1.1节中描述。在反应的最后激...

Watch this Scientific Journal Video about Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay at JoVE.com

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

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

quantification-bacterial-histidine-kinase-autophosphorylation-using

Research

JoVE Journal

Biochemistry

8.5K 次观看.  Stony Brook University. We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

视频: Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Assaying Protein Kinase Activity with Radiolabeled ATP

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

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

用放射性标记的ATP测定蛋白激酶活性

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

科研

生物化学

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

开发基于点击的检测方法，用于筛选 HAT1 酶活性

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

摘要

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此视频中的章节

用放射性标记的ATP测定蛋白激酶活性

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

衡量可逆的生物素的血浆膜蛋白吞价格

用于蛋白质磷酸化定量的优化单分子下拉测定

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法