这项工作是由加拿大健康研究协会（CIHR）（澳门币97774），自然科学和工程研究理事会，加拿大（NSERC，批NR：480619）。 KS是新研究者奖1 KRESCENT（加拿大，加拿大肾脏病学会和加拿大卫生研究院的肾脏基金会联合奖）和安大略省的研究和创新，从早期的研究者奖的收件人。 fw是由李嘉诚奖学金支持。

1。 大肠杆菌的转型大肠杆菌与PGEX-RhoA蛋白（G17A）结构<ol><li>准备LB琼脂溶解在100毫升DH 2 O 2.5克LB和1.5克琼脂高压灭菌和冷却估计50-55°C，这作为一个经验法则，是当烧瓶可以舒适举行。 </li><li>准备氨苄青霉素（Amp）的股票，通过溶解在DH 2 O 50毫克/毫升注射器过滤器和冻结闲置等分。从1.1 LB琼脂加入100安培股票μL（终浓度为50微克/毫升）。涡流混合，倒入10厘米细菌菜（15-20毫升/皿）。允许它巩固（15-30分钟）和4未使用的板倒°C间储存为2-3周。 </li><li>改造E。大肠杆菌 ，快速解冻DH5α感受态细胞分装在冰浴。新增1μlpGEXRhoA（G17A）稀释至25-50 ng /μl的DNA的。轻弹管混合并在冰上孵育30分钟。在42℃热休克45秒和地方上冰2分钟。新增900万亩，L SOC培养基，用颤抖的增长在37°C一小时</li><li> 50-100μl转化细菌的传播LB琼脂放板使用巴斯德吸管弯曲无菌。在37℃培养箱中孵育板右侧5分钟，然后反转和成长过夜。 </li><li>板准备的GST标签蛋白（步骤2.1）将有所回升，从一个单一的殖民地。以供将来使用，包装和储存板倒在4°C，约3个星期。此外，细菌的股票，可以更长时间的存储准备在2毫升无菌的LB放大器在37°C过夜，用颤抖的日益增长的个人殖民地。混合等分无菌甘油以1:1的比例，并冻结80％-80°C。 </li></ol> 2。制备GST-RhoA蛋白（G17A）珠<ol><li>准备加入25克磅到1升卫生署2 0和灭菌LB。当冷却，加入50μl的放大器，从股票到50毫升LB（50μg/ ml的最终浓度）。接种与一个孤立的山坳ony转化细菌和增长在37°C，搅拌过夜。当全密度（OD值600&gt; 1.0）稀释450毫升LB放大器和一个额外的30分钟增长在37°C。 </li><li>准备通过溶解在10毫升DH 2 O 0.238Ğ100毫米原液的异丙基BD-硫代半乳糖苷（IPTG）在分装储存于-20°C诱导细菌产生Rho蛋白加入500μLIPTG的100毫米至500毫升的文化（终浓度为100μm）。温度降低到22-24℃，成长为〜16 H小时。 </li><li> 10分钟的自旋文化在4°C，在3600Ğ如果需要的话，500毫升的文化可分为50毫升离心管。冻结颗粒（S）至少1小时（或最好过夜），在-80°C。 </li><li>准备200毫升裂解液中含有20毫米肝素钠（0.95克）/ pH值7.5; 150 mM氯化钠（1.75克）; MgCl 2的 5毫米（0.203克），1％TX-100（2毫升）。准备1M DTT（1.542克10毫升DH 2 O的股票解决方案）和100毫米PMSF（0.174 g/10毫升乙醇）。准备裂解液+，补充10毫升为1mM DTT（10微升）的股票和1毫米PMSF（100μL的股票）和一个完整的迷你蛋白酶抑制剂片。 </li><li>冰工作，加入裂解液10毫升+从步骤2.3颗粒。悬浮彻底温和涡旋和吸取。避免起泡。超声波清洗1分钟，有50％的脉冲4冰。在15,000-20,000Ğ旋转声振裂解液在4°C 15分钟，取出澄清的超声粉碎（上清）无菌管上限15毫升。 </li><li>准备轻轻含有75％到15毫升管的泥浆和转移335μL混合原管谷胱甘肽琼脂糖凝胶。使用大口径的尖端吸取珠。加入10毫升冷PBS，旋转500克为5分钟，在4°C。弃上清，加入1 mL裂解液+珠和自旋为以前的洗。弃上清，加入裂解液+，使50％的泥浆。 </li><li>加入250μL的角球从2.5步上清ibrated珠浆。在4°C旋转45分钟。 </li><li>准备500毫升哈佛商学院，含有20毫米肝素钠（2.38克）/ pH值7.5和150 mM氯化钠（4.38克）在DH 2 O准备1M氯化镁2 DH 2 O的 10毫升（0.952克）和1M DTT（1.542克）10毫升DH 2股票解决方案。准备哈佛商学院+，补充100毫升，只是在使用前用5毫米MgCl 2的现货（50μL）和1 mM DTT（100μL股票）。 </li><li>旋转5分钟从2.7步珠在4°C，500克倒掉上清，洗珠2X裂解液+ 10毫升，10毫升沸+和2倍。最后一次洗涤后重新悬浮在哈佛商学院的珠子+辅以BD BaculoGold蛋白酶抑制剂（20μL50X的BD BaculoGold /毫升），使50％的料浆。 </li><li>稀释10μl2X Laemmli样品含有β-巯基乙醇的缓冲准备最后的珠子的。使牛血清白蛋白（BSA）的标准。使用2毫克/毫升股票（BSA的0.02克10毫升DH 2 O）的。 10μL股票混合10微升的Laemmli（最后20微克）; 5μL5μLDH 2 O和10μL的Laemmli（最后的10微克）的股票;和2.5微升7.5μLDH 2 O和10μL的Laemmli股票（最后5微克）。煮沸所有样品（5分钟）。旋珠样本和运行上清与BSA的标准和10％的SDS-聚丙烯酰胺凝胶分子量标记。 </li><li>在10毫升醋酸，40 mL甲醇和50毫升DH 2准备（0.1克）和脱色液（500毫升，400毫升的甲醇和醋酸100毫升DH 2 O的 ）Comassie蓝染色。在室温下储存。凝胶染色20-30分钟，去除染料（可重复使用多次）和脱色液冲洗两次。继续脱色轻轻摇动几个小时，直到乐队都清晰可见。 </li><li>估计耦合珠BSA的标准作为参考（ 图2）使用浓度的GST-RhoA蛋白（G17A）。 aliquot的等量含1.5毫升微型离心管〜10-15微克蛋白珠。商店珠在4°C，在一天之内使用。冻结在-80°在哈佛商学院的C + + /甘油以3:1的比例，在几天之内使用。 </li></ol> 3。全球环境基金Pulldown分析核苷酸RhoA蛋白（G17A）珠<ol><li>增长在10厘米的菜汇合的细胞。血清剥夺至少3个小时的治疗需要。 </li><li>在步骤2.4准备裂解液+。准备足够的裂解液700μL/碟，再加上一些额外的金额，以便吸取错误。使用前加入蛋白酶抑制剂。 </li><li>冰工作，从菜培养基，用冰冷的哈佛商学院。删除所有的哈佛商学院和700μL裂解液+添加到每一道菜。旋流板，涵盖所有领域，刮去，并收集到编号为1.5毫升管裂解。自旋为1分，在4°C 15,000Ğ将用于检测上清。 </li><li>如果你的手机号码是相当于在所有菜被测试，你可以省略做蛋白检测，并移动到步骤3.5。否则每个使用Bio-Rad公司的快速蛋白测定上清液蛋白浓度测量和均衡数量和浓度上清液。总蛋白量取决于（通常1-1.5毫克LLC-PK1细胞蛋白）的细胞类型。 </li><li>取出30μL2X减少Laemmli样品缓冲液，煮沸30μl每个上清和混合的步骤3.7作废。剩余的上清加入GST-RhoA的珠（G17A）从步骤2.12等分。 45分钟旋转4°C。 </li><li>旋转10秒珠在4°C，在6800Ğ弃上清，珠裂解缓冲液洗3倍，在同样的方式之间的洗涤纺纱。彻底清除最后洗净，用1毫升注射器装上了地下30针，并添加20μL的2X减少Laemmli样品缓冲液。煮沸5分钟。旋转球珠和运行上清立即（最好）或存储它ţ-80℃，以后进行分析。 </li><li>运行20μL总细胞裂解物对你正在研究全球环境基金的规模适当比例的SDS-聚丙烯酰胺凝胶和沉淀的蛋白质样品。通过Western印迹使用特异性抗体检测您所选择的全球环境基金。 </li></ol> 4。代表结果该协议第1部分和2描述GST-RhoA蛋白通过SDS-PAGE（见协议大纲图1），再加上谷胱甘肽琼脂糖珠和其测试（G17A）的准备。一个典型的考马斯染色凝胶上图2所示。与洗脱的蛋白质样品应包含在约50 kDa的（ 图2，泳道6）单波段。估计该蛋白的浓度可使用BSA参考样品。在图2的例子中，RhoA蛋白（G17A）浓度估计为15μg/10μL。因此，10μL/管分装准备。典型的产量<html>在我们的手是从10毫升细菌裂解15-20微克蛋白。该协议的第3部分介绍了亲和沉淀法（见概述图1）。一个成功的全球环境基金的检测，检测激活的交换因子GEF-H1的图3所示。 RhoA的蛋白（G17A）抓获一些GEF-H1的对照组（未处理）细胞裂解，表明GEF-H1的基底活动。但是金额沉淀在细胞增加与炎性细胞因子肿瘤坏死因子-α（TNF-α）治疗，肿瘤坏死因子-α激活GEF-H1的5,7与概念一致。重要的是，总的细胞裂解类似GEF-H1基因在控制和治疗的样品量，暗示治疗，并没有改变GEF-H1的水平，并在实验中使用的输入是平等的。 <img alt="图1" src="/files/ftp_upload/3932/3932fig1.jpg" /> 图1。协议的概述。 ove_content“&gt; <img alt="图2" src="/files/ftp_upload/3932/3932fig2.jpg" /> 图2。代表珠准备协议的结果。一个成功的GST-RhoA蛋白（G17A）珠准备考马斯染色凝胶。使用10％的丙烯酰胺凝胶的SDS-PAGE分离珠样品和BSA蛋白标准。为了测试的珠子，10μL含有GST-RhoA蛋白（G17A）最后珠浆稀释减少Laemmli样品缓冲液和水煮5分钟1:1。珠纺简要上清液装入凝胶。被装在下面的示例：1巷：分子量（兆瓦）（FroggaBio BLUeye预染蛋白质梯）;泳道2-4：牛血清白蛋白（BSA）5，10和20微克; 5巷空巷6：10 μL新鲜配制的RhoA蛋白（G17A）珠。完成分离后，凝胶用考马斯蓝染色后脱色，揭示蛋白质。 GST-RhoA的蛋白（G17A）的分子量约为50<html> kDa和运行约48 kDa的标记水平。在这个特别的样品GST-Rho蛋白的浓度据估计，约15μg/10μL浆。 <img alt="图3" src="/files/ftp_upload/3932/3932fig3.jpg" /> 图3。代表GEF激活作用的检测显示TNF-α诱导活化的GEF-H1。 5分钟与10 ng / ml的TNF-α的融合的LLC-PK1细胞治疗。治疗后的细胞裂解和活跃全环基金使用的GST-RhoA蛋白（G17A）约束珠被抓获。使用反GEF-H1的抗体（Cell Signaling公司）与西方印迹检测沉淀蛋白质（顶部印迹）和总细胞裂解样品（底部印迹）GEF-H1的存在。请注意在相对等效输入与非治疗细胞TNF-α治疗的沉淀GEF-H1的增加额，说明一个成功的结果。 3932/3932fig4.jpg“/&gt; 图4。一个“糟糕的结果”。虽然全球环境基金下拉检测导致一些珠拍摄的GEF-H1的，金额从控制沉淀和TNF-α处理的细胞是相同的。因此，肿瘤坏死因子-α，知道在这种情况下，激活了GEF-H1基因没有诱导激活。随后的故障排除在这个特殊的实验表明，TNF-α的使用是不是够新鲜，可能退化。

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T., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Lsc/p115+RhoGEF+and+LARG+in+regulating+RhoA+activity+downstream+of+adhesion+to+fibronectin.">A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin.</a> J. Cell Sci. 120, 3989-3998 (2007).</li><li>Arthur, W. T., Ellerbroek, S. M., Der, C. J., Burridge, K., Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPLN,+a+guanine+nucleotide+exchange+factor+for+RhoA+and+RhoB,+but+not+RhoC.">XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.</a> J. Biol. Chem. 277, 42964-42972 (2002).</li></ol>

作者没有透露。

这里介绍的方法是唯一可用的激活全环基金，可以按照细胞中环基金的积极池非放射性检测。该法是类似以下的小GTP酶的激活以及对Rac和Cdc42全环基金用于检测的降水。这些实验使用不同的GST标签蛋白，并从此处所述的略有差异，但基本步骤是相同的​​。因此，这个协议可以很容易地被改编为其他小GTPase和GEF激活检测。 最近提出的全球环境基金法修改为9,10核分数申请。随着进一步的修改，在细胞内的其他车厢的GEF激活的测试可能也是可能的。 我们使用该方法的研究激活上皮细胞系5,6,7全环基金。随着一些优化，此法应该是足够的，以检测任何细胞株的全环基金。当adapti吴到一个特定的细胞类型，找到最佳的细胞数量，裂解液量，为全球环境基金和检测方法进行测试（一个很好的抗体免疫印迹很重要）。检测的初始设置，最好是使用一个被称为激活全球环境基金的利息刺激。使用一个未知的刺激时，总是用阳性对照，以验证您的检测工作。此法可用于检测Western印迹称为全环基金的激活。然而，这也足以识别未知的全环基金。对于这一点，从控制和刺激的样本捕获的全环基金应在考马斯染色凝胶分析。只有在刺激的样本中出现的乐队可能包含激活的全环基金，可用于识别发送质谱（如5）。 最后，一​​个警示说明。该法的基础上，使全环基金活跃的翻译后修饰细胞裂解后保留的假设。事实上，这显然是CA本身为全环基金的数量，因为它的机构，此法已用于各组检测激活不同的全环基金，包括全球环境基金H1，p115RhoGEF和XPLN（如5,11,12,13）。保存的活跃状态，但是可能不会发生在所有全环基金的情况下，因此，可以想象的是，此法不适用于所有的全环基金。它也必须指出，许多环基金发挥对多个小GTP酶的活性。因此，当一个特定的全球环境基金研究，建议，以配合此法与其他小GTP酶，以及研究RhoA蛋白，Rac1和Cdc42功能研究全球环境基金沉淀检测。 在协议中的关键步骤： 转化细菌菌落应挑选新鲜，适当的准备板，放大器，良好的生长和产量，以确保足够的选择。从其他来源获得的细胞转化的条件可能会有所不同，应谘询。 </p&gt; 在4°C水冷解决方案和离心机，应执行协议（从2.3步）的蛋白制备和检测（步骤3.2）的所有步骤。 为了获得均匀的悬浮细菌裂解（2.5步）应该全面和完整的。当裂解细菌，旋涡和吸取裂解交替，同时保持在4°C，并确保超声冰，以防止蛋白质变性。如果使用不同的sonicator模型，条件可能需要进行调整。超声粉碎的珠子孵化应始终在4°C​​，在肩，以确保足够的约束力，并应小心保持时序一致。 GST-RHO突变有些不稳定时，在细菌中表达，所以最好使用马上或在几天内准备珠。 沉淀法（第三部分）是时间和温度敏感，的主动全环基金可以很容易地从细胞裂解物丢失，这样的步骤，应尽快进行。 以下部分包含了一些故障排除技巧。 没有或突变，在最后的珠子准备RHO蛋白量非常低，这可能会造成低效的诱导，细菌裂解不足，或蛋白质的损失在筹备过程中或存储。为了帮助解决这些可能性，细菌样本进行分析诱导前后。如果是穷人诱导的蛋白质重复使用一个殖民地，从一个新鲜的条纹板或再能干细胞转化的过程。不同的IPTG浓度和诱导时间也应该进行测试。如果裂解不足（即蛋白质仍然代替上清丸）不同的裂解液中的盐和洗涤剂浓度可以尝试。替代超声倍和应考虑设置，前后超声的样品可以通过显微镜检查，以确定裂解效率。 无沉淀，全球环境基金，即使GST蛋白是目前珠：这可能是由于技术问题，在沉淀法，或缺乏真正的研究全球环境基金的使用施加的刺激激活。始终使用已知的刺激作为阳性对照，以验证您的检测工作。在几天之内准备的珠子。如果沉淀法捕捉不到全球环境基金的研究在各种条件下的金额，确认您的全球环境基金是目前是被用来检测（步骤3.3），离心后上清检测。确保所有缓冲区和蛋白酶抑制剂是新鲜的，和冰尽可能快地执行所有步骤。增加输入蛋白量（例如，通过使用从2板/样品的裂解）。如果沉淀实验结果显示，基底降水tation您的全球环境基金，但无明显差异之间的控制和刺激的样品（ 图4），通过验证，应用的刺激工作使用其他已知的影响（例如通过检测的其他信号通路的激活）开始排除故障。考虑改变治疗条件和/或浓度。从文献报告的数据，如何刺激激活RHO或其他信号来预测可能的时间和浓度依赖。优化治疗时间时，使用短期和长期的时间点，作为全球环境基金的激活可能是最好的检测时间点之前以及检测RHO激活。最后，相同的刺激可能会导致不同程度的激活因在细胞反应的变化，引起通道数，细胞汇合，等

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> PGEX-RhoA蛋白（G17A） </td><td> addgene </td><td>即将推出</td><td>产生在实验室基思博士Burridge（北卡罗莱纳大学）（3号） </td></tr><tr><td> DH5α， </td><td> Invitrogen公司</td><td> 18258-012 </td><td>存储-80°C间</td></tr><tr><td> SOC培养基</td><td> BioShop </td><td> SOC001.10 </td><td></td></tr><tr><td>的LB </td><td> BioShop </td><td> LBL407.1 </td><td>保持无菌</td></tr><tr><td>甘油</td><td> BioShop </td><td> GLY002.1 </td><td></td></tr><tr><td>氨苄青霉素</td><td> BioShop </td><td> AMP201.25 </td><td>股票贮存于-20°C </td></tr><tr><td>洋菜</td><td> BioShop </td><td> AGR001.500 </td><td></td></tr><tr><td>经IPTG（ISO丙基BD-thiogalacto的，pyranoside） </td><td> calbio-化</td><td> 420322 </td><td>储存在-20°C原液</td></tr><tr><td>谷胱甘肽琼脂糖珠</td><td> GE医疗集团</td><td> 17-0756-01 </td><td></td></tr><tr><td> PBS 10倍</td><td>西格玛</td><td> D1408 </td><td></td></tr><tr><td> HEPES </td><td>西格玛</td><td> H4034 </td><td></td></tr><tr><td>氯化钠</td><td> BioShop </td><td> SOD001.10 </td><td></td></tr><tr><td> MgCl 2的 </td><td>西格玛</td><td>的M-9272 </td><td>只是在使用前</td></tr><tr><td> TX-100 </td><td> Sigma-Aldrich公司</td><td> X100 </td><td></td></tr><tr><td>数码地面电视</td><td> OmniPur电解二氧化锰</td><td> 3860 </td><td>只是在使用前</td></tr><tr><td> PMSF </td><td>西格玛</td><td>的P-7626 </td><td>只是在使用前</td></tr><tr><td>蛋白酶抑制剂50X </td><td>屋宇署Baculo的黄金</td><td> 51-21426Z </td><td>只是在使用前</td></tr><tr&gt; <td>完整的迷你，无EDTA的10倍</td><td>罗氏公司</td><td> 11 836 170 001 </td><td>只是在使用前</td></tr><tr><td> Laemmli样品缓冲2X </td><td> BIO-RAD </td><td> 161-0737 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>西格玛</td><td> M7154 </td><td>只是在使用前</td></tr><tr><td>考马斯亮蓝</td><td> BIO-RAD </td><td>的R-250 </td><td></td></tr><tr><td>醋酸</td><td>卡利登</td><td> 1000-1 </td><td></td></tr><tr><td>甲醇</td><td>卡利登</td><td> 6701-7 </td><td></td></tr><tr><td> BIO-RAD蛋白质含量</td><td> BIO-RAD </td><td> 500-0114 </td><td></td></tr><tr><td> BLUeye阶梯</td><td> FroggaBio </td><td> PM008-0500 </td><td></td></tr></tbody></table> 表1。试剂使用。 <table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td&gt; <td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>第RC-5B离心机</td><td> sorvall </td><td> SS-34转子</td><td>使用在4°C间</td></tr><tr><td>离心分离</td><td> sorvall-Thermo Scientific的</td><td> ST-16R </td><td>使用在4°C间</td></tr><tr><td>微型离心机</td><td> Eppendorf公司</td><td> 5417R </td><td>使用在4°C间</td></tr><tr><td>细菌摇床</td><td> INFORS羟色胺Ecotron </td><td>公司的CH-1403 Bottmingen </td><td>使用在37°C或22°C间</td></tr><tr><td> sonicator </td><td>布兰森</td><td> sonifier-450 </td><td>在室温下使用</td></tr><tr><td>肩</td><td> GLAS-COL </td><td> 099A CR4012 </td><td>使用在4°C间</td></tr></tbody></table> 表2。使用的专用设备。

affinity precipitation of active rho-gefs using a gst-tagged mutant rho protein (gst-rhoa(g17a)) from epithelial cell lysates

Rho家族小GTP酶的蛋白质是细胞骨架的中央监管机构，并控制多种细胞过程，包括细胞迁移，基因表达，细胞周期进程和细胞粘附1。 RHO蛋白质是活跃的分子开关是在GTP结合，并在国内生产总值的束缚态无效。其激活介导的鸟嘌呤核苷酸交换因子（GEF）的蛋白质家族。卢全环基金构成一个大家族，重叠的特殊性2。虽然已经有很大进步作出识别激活特定信号的全环基金，仍然有许多问题，其余有关的具体规定，这些蛋白质的途径。的Rho环基金的数量超过70，每个单元表示一个以上的全球环境基金的蛋​​白质。此外，许多这些蛋白质激活不仅RHO，但其他家庭成员，进一步促进监管网络的复杂性。重要的是，探索全环基金如何监管要求的方法，按照不同的刺激活化细胞活跃的个人全环基金池。在这里，我们提供的方法来评估和量化提供积极具体的Rho-环基金使用亲和沉淀法一步一步的协议。此法是几年前开发Burridge实验室3,4和我们用它在肾小管细胞株5,6,7。该法的“核苷酸自由”突变RhoA的优势，为活跃的全环基金的高亲和力。突变（G17A），使蛋白质无法结合GDP或GTP和这个国家模仿的中间状态，则势必对全球环境基金的。 GST标签的版本的这种突变蛋白表达和纯化从E.大肠杆菌 ，势必谷胱甘肽琼脂糖珠和沉淀活跃未处理和刺激细胞裂解全环基金。作为最全环基金被激活，通过翻译后修饰或释放抑制绑定，他们的活动状态被保留在细胞裂解物，他们可以通过检测此法8。捕获的蛋白质可以被探测已知的全环基金通过检测特异性抗体，用免疫印迹，或通过质谱分析，以确定某种刺激激活的未知全环基金。

Watch this Scientific Journal Video about Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates at JoVE.com

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

这里介绍的方法检测，按照激活RhoA的具体GDP / GTP交换因子（全环基金）在培养细胞中的突变RhoA的GST融合蛋白具有高亲和力激活全环基金的使用。全环基金沉淀的细胞裂解物，免疫印迹和密度定量检测。

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

affinity-precipitation-active-rho-gefs-using-gst-tagged-mutant-rho

Research

JoVE Journal

Biology

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

16.5K 次观看.  St. Michael's Hospital. 这里介绍的方法检测，按照激活RhoA的具体GDP / GTP交换因子（全环基金）在培养细胞中的突变RhoA的GST融合蛋白具有高亲和力激活全环基金的使用。全环基金沉淀的细胞裂解物，免疫印迹和密度定量检测。

视频: Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

科研

生物学

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

转基因检测鼠类量化男性生殖细胞突变频率

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...