Name: assaying protein kinase activity with radiolabeled atp
Uploaded: 2017-05-26T14:00:00.000+00:00
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作者感谢Cobb实验室的所有现任和前任成员有价值的工作和讨论，Dionne Ware提供行政协助。这些研究得到了National Institutes of Health Grant R37 DK34128和Welch Foundation Grant I1243对MHC的支持

激酶纯化资源和一般免疫沉淀管道注意:用于该测定的激酶可以来源于培养细胞的免疫沉淀物，或通过重组方法如亲和标记的纯化6 。以下是标记免疫沉淀的通用方案，其可能必须根据感兴趣的激酶进行修饰。一切尽可能保持在冰上。 <ol><li>向200μL细胞裂解液（通常〜1 mg / mL总蛋白浓度）中加入2μL1 mg / mL抗体，并在4℃下摇摆孵育1小时。 </li><li> 通过在5,000 xg（"触摸旋转"）下在4℃下短暂旋转30秒至1分钟，将蛋白质琼脂糖珠洗涤2-3次，并用移液管除去上清液，并将缓冲液重悬于缓冲液。 <ol><li>制备裂解缓冲液:50mM HEPES pH 7.7,150mM NaCl，1.5mM MgCl 2，1mM EGTA，10％甘油，0.2mM原钒酸钠，100mM氟化钠，50mMβ-甘油磷酸酯，0.1％Triton X 100或0.1％NP-40。 </li></ol></li><li>将30μL50％的蛋白A琼脂糖凝胶珠浆液在裂解缓冲液中加入到裂解物中;在4℃下孵育1小时，同时摇摆。 </li><li>在4℃下触摸旋转以沉淀珠并除去上清液。 </li><li>用1mL珠洗涤缓冲液（1M NaCl，20mM Tris pH 7.4）洗涤3次。 </li><li>用1x激酶反应缓冲液（10mM HEPES pH8.0,10mM MgCl 2 ）洗涤一次。去除尽可能多的缓冲液，而不去除珠子。 </li></ol> 2.初始化激酶反应注意:对于IP激酶测定，〜15μL的悬浮珠是典型的起始样品。对于重组蛋白激酶测定，典型的起始量范围为1-10至25-50μL最终反应体积中的1-10-1.0μg。调整重组蛋白质样品的体积s等于其在初始化测定之前存储的缓冲液。当使用大量的激酶时，包括载体蛋白如BSA以帮助在测定期间保持蛋白质的稳定性。 <ol><li> 准备下面给出的5x激酶反应缓冲液: 50mM HEPES pH 8.0 50mM MgCl 2 50mM苄脒（蛋白酶抑制剂） 50mM DTT（还原剂） ATP（未标记或"冷"）250μM </li><li> 将激酶样品保存在1.5 mL管中。将以下反应混合物在分开的冷却的1.5mL管中制备: 21μLH 2 O 6μL5x激酶反应缓冲液 1μL放射性标记（"热"）ATP 2μL底物（〜5 mg / mL） 注意:对于重组激酶测定，可以调节体积以适应纯化的蛋白质。计算使最终反应体积为30μL。使用这个配方准备一个主人混合。在加入标记的ATP之前，将H 2 O稀释至0.01mCi /μL的比活性，然后加入反应混合物中。 </li><li>为了初始化测定，将整个反应混合物加入激酶样品中，并根据测定的激酶的活性在30℃孵育5分钟至1小时。 注意:当使用其活性尚未测定的激酶时，在时间点之间以5分钟间隔进行激酶活性的时间过程。 </li></ol> 3.反应终止和SDS-PAGE <ol><li>放入冰上停止反应，并加入7.5μL5x Laemmli样品缓冲液6 。 </li><li>在热块中在100℃下加热30秒至2分钟。 </li><li> 在10-15％SDS-PAGE凝胶上，每孔接触20μL/孔。运行凝胶足够长以分离激酶和底物（通常在5 W的恒定功率下1小时）确保保持凝胶装置屏蔽以限制暴露于32 </suP&gt; P上。 <ol><li>在这里，使用自制的凝胶装置，但标准的市售迷你凝胶是完全适合的。使用尺寸如下: 凝胶:8厘米宽，5.5厘米长（分辨），1.5毫米厚度。 梳子:15孔，1.5mm厚，2.9mm宽，16mm深</li></ol></li></ol> 4.凝胶染色和干燥注意:对于所有步骤，请务必使用放射性屏蔽和佩戴个人防护装备来减少32 P的个人暴露。有关具体步骤的更多信息，包括一些有用的参考。 <ol><li>从玻璃/氧化铝平板上取出凝胶，并置于50毫升考马斯染色剂（10％冰醋酸，50％甲醇，0.25％R-250染料）中1小时。对于该步骤，使用比凝胶本身略大的容器。 8 </li><li>将凝胶从染色剂移至定影液（10％冰醋酸）d，20％甲醇）以去污。将凝胶在600-700mL定影液中以50rpm在轨道振荡器上摇动过夜。将泡沫或打结的实验室擦拭物放在容器中，用凝胶吸收考马斯染料8,9 。 </li><li>从固定溶液中取出凝胶，并在温和搅拌下浸泡在200 mL甲醇中1-2分钟，直到凝胶变成乳白色。这将有助于防止干燥步骤中的裂纹。 </li><li>用甲醇将14厘米x 14厘米的定性滤纸弄湿，放在平板凝胶真空干燥器上。将凝胶放在滤纸正面朝上。 </li><li>小心地用塑料盖住凝胶，以避免皱纹和气泡。在80℃下运行干燥器1.5小时。在真空得到打开盖子之后，如果需要的话，用软橡胶支架将所有的气泡推出。 </li></ol>放射自显影和闪烁计数<ol><li>清除dri并将凝胶上的滤纸上贴上自动标记，磷光标尺或点。如果使用点确保它们处于非对称模式。这将使曝光和显影后的凝胶与胶片保持一致。 </li><li>使用盖革计数器检查信号强度。对于较弱的信号，在-70°C至-80°C的强度屏幕曝光会增加胶片带密度。 </li><li>在黑暗的房间里，将干凝胶放置在胶片盒中，胶片和增强屏幕从上到下按顺序依次为:凝胶，薄膜，增强屏幕。 </li><li> 将增强屏幕连接到盒子的盖子上，并将胶水贴在适当的位置，使此步骤更容易。当从32P的β粒子照射时，增强屏幕发光。这些光辐射比β粒子更有效地穿透膜。 <ol><li>确保从增强屏幕发出的波长相应池塘到电影最敏感的波长。 </li><li>使用盖革计数器确定多长时间才能使用曝光:如果cpm计数为〜100或更低，请在-70°C下过夜。如果计数为〜10,000，则从1小时开始曝光并从那里进行优化。 </li></ol></li><li>在曝光结束时，去除胶片并在医疗/ X光片处理器中发展。如果在-70至-80℃下进行曝光，则可以将盒子温热至室温，以尽量减少膜上的凝结，或者在冷凝液形成10之前立即取出膜。 </li><li>将膜放在干燥的凝胶/滤纸上，并将标记/点的图像与干燥凝胶上的标记/点对齐。在电影上标记对应于蛋白质标准的条带。标记蛋白质标准和哪些通道的反应是将来参考。 </li><li>从胶片中消除与胶片上感兴趣的条带相对应的胶带，并将其放置在7mL闪烁瓶中。加入4 mL闪烁液，用液体闪烁计数器计数。确认计数器设置为监视32 P的正确能谱窗口。 注意:确保从无激酶对照泳道切除对应于底物的条带。这将用于计算将从所有样品闪烁计数中减去的背景辐射。 </li></ol> 6.结果分析注意:放射自显影图提供了结果的定性可视化。为了精确定量，可以用闪烁计数器测量32 P掺入。数据通常用相对活动来表示， 如图1B所示 。只要维持所有样品的均匀条件，比活度的相对测量就足以比较治疗。 <ol><li>当计算绝对比活动值时，请执行严格的操作对所有反应条件（包括激酶，底物和冷ATP浓度）进行优化，以确保在一段时间过程中活性的线性范围（ 例如 2×[激酶] = 2×比活性）。对于具有高比活性的激酶，可以进行具有增加的底物浓度的测定，因为激酶活性受底物量的限制。 </li><li> 以每分钟每mg激酶转移的磷酸纳米微粒单位计算感兴趣的激酶的比活性。 <ol><li>从计数带中的cpm中减去空白。 </li><li>计算热ATP的衰减:[（1/2）^（t / t 1/2 ）] x 0.95其中t是参考日期以后的天数（应由供应商提供）， t 1/2是32 P的半衰期为14.3天，0.95反映了闪烁计数器的效率。示例:如果使用28天的热ATP，则衰减值应为0.245。 </li><li>计算器测定中总ATP的皮摩尔（pmol）（通常等于反应中冷ATP的量）:测定体积（μL）×[冷ATP]（μM）=总ATP（pmols）。实施例:30μL×50μM= 1500pmol </li><li>计算cpm / pmol ATP:[热ATP x（2.2×10 7 ）×衰变系数] / pmol的冷ATP。 注意:2.2 x 10 7的值将0.01 mCi /μL的ATP转换为cpm。 </li><li>以mg计算测定中的激酶量。对于重组激酶和免疫沉淀的激酶，标准方法如BCA测定适用于测定起始浓度。实施例:在50μL激酶反应中，wtERK20.0002μg/μL为0.01μg，或1×10 -5 mg。 </li><li>计算测定中的总cpm。组装30μL反应，每个反应在凝胶上运行20μL。将计数的cpm（空白减去后）乘以总测定体积/体积负载的因子。继续以上例如，将所有计数乘以1.5或30/20。 </li><li>计算测定的激酶的比活性: 测定中的总cpm）/（cpm / pmol ATP）/（测定时间（分钟））/（激酶的量，以mg计） 注意:将上述值除以1,000，可得到纳摩尔/分/毫克的比活度</li></ol></li><li> 计算多少摩尔磷酸盐掺入底物（只要其分子量已知）。一旦反应完成，这用于确定特定蛋白质上磷光体的数量。 测定中的总cpm）/（cpm / pmol ATP）] /（pmols中的底物量） </li></ol>

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	<li>Raman, M., Chen, W., Cobb, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+and+properties+of+MAPKs.">Differential regulation and properties of MAPKs.</a> Oncogene. 26 (22), 3100-3112 (2007).</li><li>Wellbrock, C., Arozarena, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Complexity+of+the+ERK/MAP-Kinase+Pathway+and+the+Treatment+of+Melanoma+Skin+Cancer.">The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer.</a> Front Cell Dev Biol. 4, 33 (2016).</li><li>Yang, T., Terman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+PKA-Mediated+Phosphorylation+of+Plexin+Using+Purified+Proteins.">Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.</a> Methods Mol Biol. 1493, 147-159 (2017).</li><li>Ross, H., Armstrong, C. G., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+method+for+the+assay+of+many+serine/threonine-specific+protein+kinases.">A non-radioactive method for the assay of many serine/threonine-specific protein kinases.</a> Biochem J. 366 (Pt 3), 977-981 (2002).</li><li>Akita, S., Umezawa, N., Kato, N., Higuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array-based+fluorescence+assay+for+serine/threonine+kinases+using+specific+chemical+reaction.">Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.</a> Bioorg Med Chem. 16 (16), 7788-7794 (2008).</li><li>Dalby, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphorylation:+Selected+Methods+in+Enzymology.">Protein Phosphorylation: Selected Methods in Enzymology.</a> J Am Chem Soc. 121 (51), 12215 (1999).</li><li>. Autoradiography Available from: <a target="_blank" href="https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography">https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography</a> (2011)</li><li>Walker, J. 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> The Protein Protocols Handbook. , (2009).</li><li>Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNK1,+a+novel+mammalian+serine/threonine+protein+kinase+lacking+the+catalytic+lysine+in+subdomain+II.">WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.</a> J Biol Chem. 275 (22), 16795-16801 (2000).</li><li>McReynolds, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+or+Mutation+of+the+ERK2+Activation+Loop+Alters+Oligonucleotide+Binding.">Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.</a> Biochemistry. 55 (12), 1909-1917 (2016).</li><li>Carlson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+discovery+of+ERK2+substrates+identifies+ERK-mediated+transcriptional+regulation+by+ETV3.">Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3.</a> Sci Signal. 4 (196), rs11 (2011).</li><li>Courcelles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+dynamics+reveal+novel+ERK1/2+MAP+kinase+substrates+with+broad+spectrum+of+functions.">Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.</a> Mol Syst Biol. 9, 669 (2013).</li><li>Myers, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+and+Hit+Validation+of+Extracellular-Related+Kinase+5+(ERK5)+Inhibitors.">High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.</a> ACS Comb Sci. 18 (8), 444-455 (2016).</li></ol>

作者没有什么可以披露的。

激酶是一种多样化的蛋白质家族，其在多种情况下已经发展了广泛的功能，激酶测定在研究几种信号蛋白方面已经非常有用，并且极大地有助于我们目前对细胞通信的理解。值得注意的是，尽管在结构和活性上存在重大差异，但使用相同的基本测定来表征两种不同的激酶。 WNK1激酶含有非典型催化口袋，临界赖氨酸转移到独特的位置，已知通过激酶和脚手架功能调节阳离子氯化物共转运蛋白的作?...

蛋白激酶是将细胞信号传递到适当的反应中所必需的复杂酶。鉴于其在维持体内平衡和预防或促进疾病状态方面的作用2 ，用于评估激酶活性的生物化学方法继续是描绘真核信号传导3的特征的有力工具。尽管使用磷酸特异性抗体的策略在测量不同治疗条件对细胞信号传导状态的影响方面已经具有高度的信息，但激酶测定允许直接根据激酶的酶活性来测量不同治疗条件的影响利益。虽然对于不使用放射性物质的类似测定法有几种选择5 ，我们仍然依靠这种方法对结果进行稳健的定量。那里是该测定的两个典型应用，两者都有价值的不同原因:免疫沉淀（IP）激酶测定（ 图1 ）和重组蛋白激酶测定（ 图2 ）。 IP激酶测定对于识别能够激活特异性蛋白激酶的因子以及测量抑制性治疗条件非常有用。简言之，将感兴趣的表位标记的激酶转染到培养的真核细胞中，进行各种处理，免疫沉淀，并测定将放射性标记的磷酸掺入模型底物（ 例如髓磷脂碱性蛋白（MBP））的能力。也可以通过免疫沉淀内源蛋白或任何数量的基因组编辑技术来进行IP激酶测定而不诉诸过表达。因为治疗是在文化中进行的，所以这种方法可以检测通过多个上游因素传播的刺激或通过体外读出的平行通路。该方法的一个主要优点是其不需要在其中直接上游或下游因子或磷酸化位点的先验知识。此外，一旦已经鉴定了感兴趣的激酶的特异性底物，与重组组分可以使用相同的激酶测定方案，以测量与天然底物的比活性，并在与质谱分析结合时鉴定特异性磷酸化位点。使用底物突变体的激酶测定可以进一步验证以这种方式鉴定的位点。最后，该方法也可用于检测和测量自磷酸化。 这里提供的方案假设用于在培养细胞中表达感兴趣的亲和标记的激酶的优化的蛋白质纯化方案或转染方法。有关转染，裂解，免疫沉淀和蛋白质纯化的更详细的论述我们建议参考Cold Spring Harbor Protocols 6 。有关测定开发和修饰的更多信息，请参阅蛋白质磷酸化:选择的酶学方法7 。

<table><tbody><tr><td>Protein A Sepharose CL-4B</td><td>GE Healthcare Life Sciences</td><td>17-0963-03</td><td></td></tr><tr><td>Radiolabeled [&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P] ATP</td><td>Perkin Elmer</td><td>NEG035C010MC</td><td></td></tr><tr><td>Laemmli Buffer</td><td>Bio-Rad</td><td>#1610737</td><td></td></tr><tr><td>Radioactive shielding</td><td>Research Products International</td><td>BR-006</td><td></td></tr><tr><td>R-250 dye (Coomassie)</td><td>Thermo Fisher</td><td>20278</td><td></td></tr><tr><td>Whatman Grade 3 qualitative filter paper</td><td>Whatman</td><td>1003-917</td><td></td></tr><tr><td>Slab gel vacuum dryer</td><td>Bio-Rad</td><td>Model 583 Gel Dryer #1651745&amp;nbsp;</td><td></td></tr><tr><td>Autorad marker</td><td>Agilent Technologies</td><td>420201</td><td></td></tr><tr><td>Phosphorescent ruler</td><td>Sigma Aldrich</td><td>R8133</td><td></td></tr><tr><td>Phosphorescent dots</td><td>Sigma Aldrich</td><td>L5149</td><td></td></tr><tr><td>Geiger counter</td><td>Ludlum</td><td>Model 3 with 44-9 detector</td><td></td></tr><tr><td>BioMax Cassette 8 x 10&quot;</td><td>Carestream Health</td><td>107 2263</td><td></td></tr><tr><td>BioMax MS Film 8&quot; x 10&quot;</td><td>Carestream Health</td><td>829 4985</td><td></td></tr><tr><td>BioMax MS Intensifying Screen 8&quot; x 10&quot;</td><td>Carestream Health</td><td>851 8706</td><td></td></tr><tr><td>Medical/X-ray film processor</td><td>Konica Minolta</td><td>SRX-101A</td><td></td></tr><tr><td>Scintillation vials</td><td>Research Products International</td><td>125500</td><td></td></tr><tr><td>Scintillation fluid</td><td>MP Biomedicals</td><td>188245305</td><td></td></tr><tr><td>Beckman LS 6500 Scintillation counter</td><td>Beckman</td><td>LS 6500</td><td></td></tr></tbody></table>

assaying protein kinase activity with radiolabeled atp

蛋白激酶能够响应于复杂的刺激阵列来控制大规模细胞变化，并且大量努力旨在揭示其调节的变构细节。激酶包括信号网络，其缺陷通常是多种形式的癌症和相关疾病的标志，使得能够操纵上游调节因子的测定平台和对于改进治疗学的研究至关重要的反应要求的验证。在这里，我们描述了一个基本的激酶测定，可以很容易地适应特定的实验问题，包括但不限于测试生物化学和药理学的作用，遗传操作如突变和缺失，以及细胞培养条件和治疗探针细胞信号传导机制。该测定使用放射性标记的[γ- 32 P] ATP，其允许定量比较和清楚的结果可视化，并且可以是mod如果用于免疫沉淀或重组激酶，特异性或典型的底物，在广泛的反应条件下使用。

WNK1 IP激酶测定将Myc标记的WNK1转染到HEK293细胞中，并用抗Myc抗体11免疫沉淀。免疫沉淀物显示对模型底物MBP以及自身的激酶活性（ 图1A ）。然后通过相同的方法测试WNK1突变体对MBP的激酶活性，此时采用GST标记的构建体（ 图1B ）。相对于野生型的突变体激酶行为的分析揭示了对于最佳WNK...

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Assaying Protein Kinase Activity with Radiolabeled ATP

蛋白激酶是对于细胞间和细胞内信号转导至关重要的高度进化的信号酶和支架。我们提出了通过使用放射性标记的三磷酸腺苷（[γ- 32 P] ATP）测量激酶活性的方案，这是一种有助于阐明细胞信号调节的可靠方法。

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 ...

assaying-protein-kinase-activity-with-radiolabeled-atp

Research

Education

JoVE Core

JoVE Journal

Molecular Biology

Biochemistry

Unit 1

Protein Function

SCIENTISTS IN ACTION

18.3K 次观看.  University of Texas Southwestern Medical Center. 蛋白激酶是对于细胞间和细胞内信号转导至关重要的高度进化的信号酶和支架。我们提出了通过使用放射性标记的三磷酸腺苷（[γ- 32 P] ATP）测量激酶活性的方案，这是一种有助于阐明细胞信号调节的可靠方法。 

视频: Assaying Protein Kinase Activity with Radiolabeled ATP

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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

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

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

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

科研

生物化学

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

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 ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

生物学

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

测量<em&gt;体外</em&gt;的酶学性质ATP酶活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

用放射性标记的ATP测定蛋白激酶活性

摘要

探索更多视频

此视频中的章节

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

寡肽竞争测定磷酸化位点测定

使用新激酶蛋白的强生化方法在分子水平上进行表征

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

含量测定的LRRK2的激酶活性在体外

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

生化分析的分析ATP依赖的染色质重塑酶活性的影响

测量

用放射性标记的ATP测定蛋白激酶活性

摘要

探索更多视频

此视频中的章节

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

寡肽竞争测定磷酸化位点测定

使用新激酶蛋白的强生化方法在分子水平上进行表征

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

含量测定的LRRK2的激酶活性在体外

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

生化分析的分析ATP依赖的染色质重塑酶活性的影响

测量

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的