这项工作得到韩国政府资助的国家研究基金会（MSIP）（第2015R1A2A1A10052663号和第2014M1A3A3A02034698号）的支持。

安全注意:本协议使用[γ- 32 P] -ATP评估AMPK的活性。磷32是放射性同位素，主要发射β辐射。由于β粒子的尺寸非常小，因此可以很容易地穿透衣服和皮肤。外部和内部暴露于β辐射可能对人体健康有害，包括引起皮肤灼伤和组织损伤。 <ol><li>执行需要使用[γ- 32 P] -ATP的所有步骤，使用适当的保护，如丙烯酸屏蔽。 </li><li>确保所有人员配备电子个人剂量计（EPD），以监测所有程序中暴露于有害辐射的程度。 </li><li>只有经过适当的培训和监管批准才能处理开源辐射。 注:在这里，所有的实验都是在首尔国立大学完成开源辐射使用培训后进行的呃和韩国政府的批准（nssc.go.kr/nssc/en）。 </li><li>根据当地法规处理放射性废物。 注:遵循首尔国立大学环境保护与安全研究所的指示。以下是美国和欧洲国家的监管机构名单:美利坚合众国，美国核管理委员会（http://www.nrc.gov/）;英国健康与安全执行（HSE）（http://www.hse.gov.uk）;法国，自由诺基亚（https://www.asn.fr/）;和德国，联邦环境，自然保护，建筑和核安全部（BMU）（http://www.bmu.de）。 </li></ol> 2. 体外竞争激酶测定<ol><li> 竞争肽的构建 <ol><li>确定由AMPK磷酸化的共有基序。 注意:AMPK识别共识基序，＃934; - [X，β] -XX-丝氨酸/苏氨酸-XXX-Φ。在共识序列中，Φ表示疏水氨基酸（ 即缬氨酸[V]，亮氨酸[L]，甲硫氨酸[M]和异亮氨酸[I]），β代表碱性氨基酸（ 即赖氨酸[K]，精氨酸[ R]和组氨酸[H]） 1 。 </li><li>根据上述共识基序从目标序列中选择丝氨酸或苏氨酸残基。使用人Nrf2的三个假定位点（＃1，包括Ser153的148-157;包含Ser335的＃2，330-339;和包含Ser558的＃3，553-562） 8 。 </li><li>商业合成模拟推定位点的10位残基肽。获得合成的产物作为每种肽的纯度大于98％的冻干粉末。 </li><li>使用激酶缓冲液溶解肽以达到71.667g / L的浓度。如果肽不溶于激酶缓冲液，则将其溶于20％DMSO。如果它是不溶性的，则模仿放置的肽可能延长现场可增加溶解度。 </li></ol></li></ol><ol start="2"><li> 体外竞争性AMPK激酶测定 <ol><li>如下制备5×激酶缓冲液:100mM HEPES，pH 7.4; 25mM MgCl 2 ; 5mM EGTA; 5mM DTT; 125mMβ-甘油磷酸酯（丝氨酸/苏氨酸磷酸酶抑制剂）; 5mM Na 3 VO 4 （酪氨酸磷酸酶抑制剂）; 0.5％（v / v）蛋白酶抑制剂鸡尾酒套装III，1mM冷ATP;和500μMAMP。 注意:AMP是测试AMPK活性的激酶缓冲液的重要组成部分。缓冲液可用于测量丝氨酸/苏氨酸激酶和酪氨酸激酶的活性。 </li><li>在冰上解冻以下重组蛋白和合成肽:AMPK; Nrf2的;和寡肽＃1，＃2和＃3。 </li><li>在冰上制备反应缓冲液:0.15μgAMPK，0.4μgNrf2（〜4pmol），0.43mg寡肽（〜300nmol）和6μL5×激酶缓冲液。一个用无菌蒸馏水（DW）将最终体积调节至30μL。 注意:在制备反应缓冲液后添加[γ- 32 P] -ATP可能会降低由于激酶自磷酸化引起的放射性信号。 </li></ol></li><li> 运行反应物 注意:以下所有过程必须使用丙烯酸屏蔽，机架或块，以及适当的个人防护设备进行保护。 <ol><li>在开始实验之前将加热块设置为30°C。 </li><li>在冰上向反应管中加入1μL[γ- 32 P] -ATP（1μCi）。通过上下移动多次混合反应缓冲液。 注意: 32 P的放射性半衰期约为14天9 。通过考虑半衰期来调节体积（ 例如，从[γ- 32 P] -ATP生产一个月后，加入4μL[γ- 32 P]-ATP使1μCi）。 </li><li>将加热块中的样品在30℃下孵育15-30分钟。在此步骤中，制备7.5％十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶。根据目标蛋白的大小调整凝胶的百分比。 </li><li>通过加入3μL10×SDS样品缓冲液（10％（v / v）甘油，20％（w / v）SDS，15.42％（w / v）二硫苏糖醇，90％（v / v） M Tris-HCl（pH6.8）和0.02％（w / v）溴酚蓝）。通过上下移动多次混合反应缓冲液。 </li><li>将样品在7.5％SDS-PAGE凝胶中在70V下运行20分钟，并在140V持续1小时。 注意:凝胶末端的溴酚蓝线含有极高的放射性信号。建议在蓝色线下方移除凝胶，然后再进行下一步以减少背景信号。 </li><li> SDS-PAGE后，从施法者轻轻取出凝胶。将凝胶放在玻璃杯中ube为下一步。 </li><li>用固定缓冲液固定凝胶20分钟（50％甲醇和10％冰醋酸）。 </li><li>将凝胶轻轻移动到滤纸上，并用透明包装纸盖上。注意，在该步骤中，凝胶可能容易撕裂。用真空凝胶干燥器在80℃下干燥凝胶1小时。 </li></ol></li><li> 可视化 <ol><li>将凝胶暴露于荧光屏或X射线胶片过夜（通常约16小时）。 注意:荧光屏在暴露于超亮的光线之后可以重复使用。当使用X光胶片时，将凝胶暴露两天。 X射线胶片具有比荧光屏更低的灵敏度。 </li><li>用荧光成像仪扫描荧光屏。将图像导出为高分辨率TIFF或位图文件。 </li><li>在可视化后，将凝胶移动到玻璃或塑料容器，以进行考马斯亮蓝（CBB）染色。 </li><li>用DW洗涤凝胶。丢弃DW并染色e用染色试剂凝胶1小时。 </li><li>弃去试剂并用DW将凝胶脱胶1小时或过夜。将凝胶放在透明塑料薄膜（或等同物）之间，用办公扫描仪进行扫描。 </li></ol></li></ol> 3.使用位点特异性突变体进行体外激酶测定<ol><li> pGEX-Nrf2的定点诱变 <ol><li>诱变引物设计 注意:设计适当的诱变引物对于诱变的成功结果至关重要。以下是设计诱变引物的一些考虑。创建一个由25到45个碱基组成的引物，在引物中间有所需的突变。引物应具有40-60％的GC含量，并终止于一个或多个G或C碱基。根据公式[T m = 81.5 + 0.41（％GC） - （675 /碱基长度） - ％不匹配），确保引物的T m等于78℃或更高。 注意:净化引物，因为其纯度对突变效率很重要。有几个设计诱变引物的网站。 <ol><li>打开"定点诱变设计引物"的引物设计程序（http://www.genomics.agilent.com/primerDesignProgram.jsp）。 </li><li>粘贴人类Nrf2 DNA序列（访问号NM_006164.4）并选择"上传翻译"按钮。 </li><li>从翻译的氨基酸序列中选择丝氨酸558残基，以更改为丙氨酸。使用诱变引物，将密码子替换为来自agc的丝氨酸至gcc，用于丙氨酸。 </li><li>考虑引物长度，T m和GC含量。 注意:诱变引物最终设计为5&#39;-ctactgaaaaaacaactc gcc accttatatctcgaag-3&#39;。 </li></ol></li><li>使用聚合酶链反应（PCR）的突变链合成反应<ol><li>在冰上准备PCR反应混合物:10×反应缓冲液，50ngpGEX-GST-Nrf2，125ng正向引物，125ng反向引物，1μLdNTP混合物（每个10mM），2.5U Pfu DNA聚合酶和无菌DW至50μL。 </li><li>用以下循环程序运行pGEX-GST-Nrf2突变体链合成的PCR:95℃30秒的1个循环;在95℃30秒，55℃1分钟，68℃90秒循环18个循环;在68℃下1个循环5分钟。 注意:根据质粒长度，循环参数可能会改变。 </li><li>将其放在冰上2分钟，冷却反应物进行下一步骤。或者，将其储存在4°C过夜。 </li></ol></li><li> Dpn I消化和突变质粒的选择<ol><li>冷却后，向每个反应物中加入1μLDpn I限制酶（10U /μL）。通过移液彻底混合并离心1分钟。 </li><li>在37℃下孵育2小时以消化亲本pGEX-GST-Nrf2双双链DNA。 </li><li>在冰上轻轻融化DH5α超感受态细胞。 </li><li>将50μL反应物加入到150μL预冷的DH5α超级感受态细胞中，并在冰上孵育至少30 min。 </li><li>将管转移到预热至42℃的加热块，并将其放置90秒。将它们放在冰上2分钟。 </li><li>向转化的DH5α细胞中加入预热的溶菌酵母（LB），并在37℃下以180rpm摇动孵育1小时。 </li><li>将转化的细胞在LB-氨苄青霉素琼脂平板上铺展并在37℃下孵育过夜。 注意:氨苄青霉素用于选择，因为质粒GEX-GST-Nrf2具有氨苄青霉素抗性标记。使用适当的抗生素，这取决于诱变质粒的抗性标记。 </li><li>从板上挑取单个菌落，将其转移至5mL LB-氨苄青霉素中，并在37℃下以180rpm摇动孵育过夜。为了验证诱变序列，评估至少三个菌落。 </li><li>使用DNA微量制备试剂盒提取DNA进行DNA序列分析;遵循制造商的协议。 </li><li>使用自动DNA序列分析仪根据制造商的方案验证诱变DNA序列。使用至少200ng的DNA构建体来验证突变序列。 </li></ol></li></ol></li><li> 重组GST-Nrf2蛋白的纯化 <ol><li>诱变GST-Nrf2融合蛋白在大肠杆菌中的表达 <ol><li>将诱变型pGEX-GST-Nrf2质粒转化到BL21细胞中。接种在含有氨苄青霉素（100μg/ mL）的25mL LB肉汤中的单个菌落，并在37℃下在旋转器上孵育过夜。 </li><li>将培养的细胞接种在含有氨苄青霉素（100μg/ mL）的100mL含有1:100稀释比的LB肉汤中。 </li><li>在37°C的温度下旋转或直到600nm处的吸光度达到约0.4-0.8（约4小时）。 </li><li>向100 mL培养细胞中加入100μL1M IPTG，制备终浓度为1 mM IPTG。 </li><li>将细胞在30℃下在旋转器上孵育6小时或过夜以进行蛋白质表达。 </li><li>以5,000×g离心细胞20分钟，并用1mL细菌裂解缓冲液（25mM HEPES，5mM EDTA，2mM DTT，0.1％CHAPS，1μg/ mL胃酶抑素，0.5μg/ mL亮抑酶肽，和1mM PMSF）。 </li><li>使用超声波均化器以最大功率输出的50％，以2秒间隔裂解细胞沉淀2分钟。 </li><li>在4℃离心细胞12,000×g 15分钟并收集上清液。 </li></ol></li><li>重组GST-Nrf2蛋白的纯化<ol><li>准备200μL的50％（v / v）谷胱甘肽 - 琼脂糖珠，并用1mL PBS（pH 7.3; 140mM NaCl，2.7mM KCl，10mM Na 2 </sHPO 4和1.8mM KH 2 PO 4 ）。通过在500×g离心1分钟收集珠子。重复洗涤步骤三次。 </li><li>将细胞裂解物加入到制备的200μL的50％（v / v）谷胱甘肽 - 琼脂糖珠中，并在4℃下在终端旋转器上孵育2小时。 </li><li>通过在500×g离心1分钟收集与GST-Nrf2蛋白结合的谷胱甘肽 - 琼脂糖珠。用31 G针头的1 mL注射器取出上清液。 </li><li>用1mL PBS洗涤珠子（pH7.3），如步骤1。 </li><li>向与GST-Nrf2组合的谷胱甘肽 - 琼脂糖珠中加入500μL洗脱缓冲液（50mM Tris-HCl和10mM还原型谷胱甘肽，pH8.0）。在4℃下在终端旋转器上孵育30分钟。 </li><li>以500×g离心1分钟并收集上清液。 </li><li>再次加入洗脱缓冲液300μL，重复步骤3.2.2.5-3.2.2.6两次。 </li></ol></li><li>纯化的GST-Nrf2蛋白的确认<ol><li>准备5μL样品，用0.5μg标准蛋白质BSA观察蛋白质数量。 </li><li>将样品在7.5％SDS-PAGE凝胶中在70V下运行20分钟，然后在140V下运行60分钟。 </li><li>用0.1％CBB染色溶液（0.1g CBB R250,40mL99％甲醇，10mL乙酸乙酸和50mL蒸馏H 2 O）将凝胶染色2小时，脱色（​​30mL甲醇， 10mL冰醋酸和60mL蒸馏水H 2 O）直到清楚显示条带。 </li><li>将脱色凝胶放在滤纸上，盖上透明包装纸。用真空凝胶干燥器在80℃下干燥凝胶1小时。 </li><li>通过体外 AMPK激酶测定的光密度测定法确定突变体GST-Nrf2蛋白的数量。 </li></ol></li></ol></li><li> 体外 AMPK活性测定与位点特异性突变体 不E:根据步骤2.2-2.4 进行体外 AMPK激酶测定，使用0.4μg纯化的野生型GST-Nrf2或没有肽抑制剂的GST-Nrf2-S558A突变体。遵循步骤1中描述的所有辐射安全准则。 <ol><li>用0.15μgAMPK，0.4μg突变体或野生型GST-Nrf2和6μL5激酶缓冲液在冰上制备反应缓冲液。用无菌DW填充30μL。 </li><li>在冰上向反应管中加入1μL[γ- 32 P] -ATP（1μCi/μL）。通过上下移动多次混合反应缓冲液并离心。 </li><li>将样品在30℃加热块中孵育30分钟。 </li><li>加入3μL10×SDS样品缓冲液停止激酶反应。 </li><li>在7.5V的SDS-PAGE凝胶中在70V下运行20分钟，然后在140V下运行1小时。 </li><li>用固定缓冲液固定凝胶20分钟，将其移至滤纸上，并用透明膜覆盖凝胶包装</li><li>用真空凝胶干燥器在80℃下干燥凝胶1小时，然后将其过夜曝光至荧光屏或X射线胶片。 </li><li>用磷光体成像仪扫描荧光屏，并根据步骤2.4将凝胶移动到可视化后的玻璃管进行CBB染色。 </li></ol></li></ol>

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	<li>Hardie, D. G., Schaffer, B. E., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK:+An+energy-sensing+pathway+with+multiple+inputs+and+outputs.">AMPK: An energy-sensing pathway with multiple inputs and outputs.</a> Trends Cell Biol. 26, 190-201 (2016).</li><li>Gwinn, D. M., 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=AMPK+phosphorylation+of+raptor+mediates+a+metabolic+checkpoint.">AMPK phosphorylation of raptor mediates a metabolic checkpoint.</a> Mol Cell. 30, 214-226 (2008).</li><li>Huang, H. C., Nguyen, T., Pickett, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Nrf2+at+Ser-40+by+protein+kinase+C+regulates+antioxidant+response+element-mediated+transcription.">Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.</a> J Biol Chem. 277, 42769-42774 (2002).</li><li>Niture, S. K., Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant-induced+modification+of+INrf2+cysteine+151+and+PKC-delta-mediated+phosphorylation+of+Nrf2+serine+40+are+both+required+for+stabilization+and+nuclear+translocation+of+Nrf2+and+increased+drug+resistance.">Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance.</a> J Cell Sci. 122, 4452-4464 (2009).</li><li>Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK-3beta+acts+upstream+of+Fyn+kinase+in+regulation+of+nuclear+export+and+degradation+of+NF-E2+related+factor+2.">GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.</a> J Biol Chem. 282, 16502-16510 (2007).</li><li>Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+analysis+by+mass+spectrometry:+myths,+facts,+and+the+consequences+for+qualitative+and+quantitative+measurements.">Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.</a> Mol Cell Proteomics. 5, 172-181 (2006).</li><li>Dephoure, N., Gould, K. L., Gygi, S. P., Kellogg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+analysis+of+phosphorylation+sites:+a+quick+guide+for+cell+biologists.">Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.</a> Mol Biol Cell. 24, 535-542 (2013).</li><li>Joo, M. S., 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=AMPK+facilitates+nuclear+accumulation+of+Nrf2+by+phosphorylating+at+Serine+550.">AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550.</a> Mol Cell Biol. 36, 1931-1942 (2016).</li><li>Sinclair, W. K., Holloway, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-lives+of+some+radioactive+isotopes.">Half-lives of some radioactive isotopes.</a> Nature. 167 (4244), 365 (1951).</li><li>Chang, B. Y., Chiang, M., Cartwright, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Src+and+RACK1+is+enhanced+by+activation+of+protein+kinase+C+and+tyrosine+phosphorylation+of+RACK1.">The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1.</a> J Biol Chem. 276, 20346-20356 (2001).</li><li>Smith, L., 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=RACK1+interacts+with+filamin-A+to+regulate+plasma+membrane+levels+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.</a> Am J Physiol Cell Physiol. 305 (1), C111-C120 (2013).</li></ol>

作者没有什么可以披露的。

作为评估由AMPK介导的预测的磷酸化位点的真实性的简单和方便的方法，在此我们描述了体外激酶测定法，其可用于使用竞争性肽发现特定磷酸化位点，并使用位点特异性突变体。从体外竞争性AMPK活性测定获得的代表性数据与使用位点定向突变蛋白的测定结果匹配，表明肽竞争测定是确定磷酸化位点的有用工具。该方法也用于鉴定由PKCδ磷酸化的特异性位点的研究，其在丝氨酸40 <sup c...

特定残基处的蛋白磷酸化在广泛的细胞过程中起着重要的作用。因此，了解信号网络需要鉴定特异性磷酸化位点。此外，磷酸化位点决定了对蛋白质功能的影响，因为蛋白质内的各个结构域具有不同的结构和功能。核因子红因子2相关因子2（Nrf2）的活性是一种主要的抗氧化转录因子，通过不同位点的磷酸化双向调节。我们的研究集中在催化Nrf2磷酸化的激酶。 Nrf2对氧化性攻击的应激反应迅速发生，主要通过丝氨酸40的磷酸化并由蛋白激酶C（PKC）-δ介导，其激活Nrf2,3,4。相反，Fyn催化Nrf2在酪氨酸5处的抑制性磷酸化68用于活动的紧密控制5 。 用于发现磷酸化位点的最常见方法是液相色谱/质谱（LC / MS）分析。可以通过这种方法生成快速高灵敏度的磷酸化位点作图数据;然而，它有几个技术限制，通常产生假阴性信号。 LC / MS分析中频繁发生序列覆盖不良。为了鉴定磷酸化位点，需要关于蛋白质最大氨基酸覆盖度的信息6 。在消化步骤期间与几种蛋白酶的目的蛋白质的蛋白水解可能有助于改善序列覆盖。识别磷酸化残基的另一个障碍是丝氨酸和苏氨酸磷酸化肽6,7经常观察到的磷酸的容易损失。不规则磷酸酯部分通常是在碎裂过程中从磷酸肽释放7 。寻找磷酸化位点时的第二个选择是使用肽微阵列法。可以使用含有衍生自感兴趣的蛋白质的肽片段的微阵列芯片筛选激酶靶位点。然而，由于微阵列芯片的生产和检测的设备要求，肽微阵列方法被认为是耗时且昂贵的。 为了克服这些挑战， 体外寡肽竞争激酶测定可用于具有已知识别基序的蛋白激酶。如果确定了激酶的共识识基序，则可以预测候选底物的推定的磷酸化位点，并且可以验证位点的真实性。这个程序最有说服力的方法是显示在其中是preicte的突变蛋白中磷酸化的消除d残基被不可磷酸化的氨基酸（ 即丝氨酸或苏氨酸转化为丙氨酸，酪氨酸至苯丙氨酸）取代。然而，突变蛋白的生产和分离是耗时的。作为研究初期的替代方案，竞争性肽激酶测定是直接和方便的。在这里，我们描述了体外肽竞争测定和磷酸化位点验证的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>208337</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Calbiochem, Nottingham, UK</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1752</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPK</td>
			<td>Upstate Biotechnology, Lake Placid, NY</td>
			<td>14-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrf2 (WT)</td>
			<td>Abnova, Taipei City, Taiwan</td>
			<td>H00004780-P01</td>
			<td></td>
		</tr>
		<tr>
			<td>[&#947;-32P]-ATP</td>
			<td>PerkinElmer Life and Analytical Sciences, Waltham, MA</td>
			<td>NEG502A</td>
			<td></td>
		</tr>
		<tr>
			<td>EZblue staining reagent</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu turbo DNA polymerase</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>600250</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>200415-51</td>
			<td>Avoid multiple thaw and freezing cycle</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Duchefa Biochemie BV, Haarlem, Netherlands</td>
			<td>L1704</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix, Santa Clara, CA</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>iNtRON Biotechnology, Sungnam, South Korea</td>
			<td>32034</td>
			<td></td>
		</tr>
		<tr>
			<td>HiYield Plus Gel/PCR DNA Mini Kit</td>
			<td>Real Biotech Corporation, Taipei, Taiwan</td>
			<td>QDF100</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bovogen Biologicals, Victoria, Australia</td>
			<td>BSA100</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4B</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>17-0756-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid glacial</td>
			<td>Duksan pure chemicals, Ansan, South Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol</td>
			<td>Daejung Chemicals &#38; Metals, Siheung, South Korea</td>
			<td>5558-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>28-9558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE kit</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-1746</td>
			<td></td>
		</tr>
		<tr>
			<td>Dancing shaker</td>
			<td>FINEPCR, Seoul, Korea</td>
			<td>CR300</td>
			<td>The machine is needed for washing step</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator/shakers</td>
			<td>N-BIOTEK, GyeongGi-Do, Korea</td>
			<td>NB-205L</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges</td>
			<td>LABOGENE, Seoul, Korea</td>
			<td>1730R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography columns</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>732-1010</td>
			<td></td>
		</tr>
	</tbody>
</table>

oligopeptide competition assay for phosphorylation site determination

特定位点的蛋白质磷酸化决定了其与其他分子的构象和相互作用。因此，蛋白磷酸化影响细胞的生物功能和特征。目前，发现磷酸化位点最常见的方法是通过液相色谱/质谱（LC / MS）分析，快速而灵敏的方法。然而，相对不稳定的磷酸酯部分通常在分裂步骤期间从磷酸肽释放，其通常产生假阴性信号。在这种情况下，使用定点突变体的传统的体外激酶测定将更准确，但是该方法费时费力。因此，使用肽竞争的替代方法可能是有利的。已经建立了5&#39;腺苷单磷酸激活蛋白激酶（AMPK）的共识识基序1 ，并使用位置扫描肽库文库验证ay 2 。因此，可以通过肽竞争测定来预测和证实用于新基质的AMPK磷酸化位点。在本报告中，我们通过说明AMPK介导的核因子红系2相关因子2（Nrf2）磷酸化来描述体外寡肽竞争激酶测定的详细步骤和程序。为了认证磷酸化位点，我们使用位点特异性突变体进行了连续的体外激酶测定。总体上，肽竞争测定提供了筛选多个潜在磷酸化位点并鉴定磷酸化位点突变体验证位点的方法。

图1和图2显示了先前报道的文献8中重复实验的结果。合成了三种模拟推定的AMPK靶位点（包括Ser153的＃1,148-157;包含Ser335的＃2，330-339;和包含Ser558的＃3，553-562）的10个残基寡肽，并将其用作在体外激酶测定。在寡肽＃3的存在下，AMPK对Nrf2的磷酸化作用大大降低（ 图1 ）。接下来，进...

Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Oligopeptide Competition Assay for Phosphorylation Site Determination

肽竞争测定广泛用于各种分子和免疫学实验。本文描述了体外寡肽竞争激酶测定的详细方法和相关的验证程序，其可用于发现特定的磷酸化位点。

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 ...

oligopeptide-competition-assay-for-phosphorylation-site-determination

Research

JoVE Journal

Biochemistry

8.3K 次观看.  Seoul National University. 肽竞争测定广泛用于各种分子和免疫学实验。本文描述了体外寡肽竞争激酶测定的详细方法和相关的验证程序，其可用于发现特定的磷酸化位点。 

视频: Oligopeptide Competition Assay for Phosphorylation Site Determination

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

科研

生物化学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

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; ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

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 ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...