这项工作得到了国家卫生研究院授予的DA-000266和医学科学家培训计划T32授权（CV，NWZ和PCS）的支持。作者还要感谢Somersault 18:24（somersault1824.com）的科学与医学插图图书馆。

1.重组HA-MOR1在培养细胞中的表达注意:按照无菌层流罩中的所有细胞培养方案。 <ol><li>用70％乙醇灭菌细胞培养层流罩，并在整个细胞培养物中保持无菌技术。 </li><li>制备人胚胎肾细胞293（HEK293）细胞培养基，补充有2mM L-谷氨酰胺，1％青霉素/链霉素和10％胎牛血清（FBS）的完全Dulbecco&#39;s修饰的Eagle培养基（DMEM）:DMEM，pH7.4 ）。 </li><li>将2.5×10 6 HEK293细胞在10毫升完全DMEM中的10厘米组织培养板上孵育，并在37℃和5％CO 2下孵育直至达到70-80％汇合（O / N）。 注意:为了收集足够的蛋白质进行完整的GTP结合实验，将至少3×10厘米的细胞平板。 </li><li>使用选择的转染试剂，用HA-MOR1转染细胞，的指导方针。孵育36-48小时。 注意:人MOR-1 cDNA（NCBI参考序列:NM_000914.4）是Gavril Pasternak的大量礼物，并被克隆到pCMV-HA中。 </li></ol>细胞分馏和膜收集<ol><li> 准备以下裂解缓冲液: <ol><li>缓冲液1:10mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），pH 7.4; 1mM乙二醇 - 双（β-氨基乙基醚）-N，N，N&#39;，N&#39;-四乙酸（EGTA）; 10％蔗糖;蛋白酶抑制剂鸡尾酒;和1mM二硫苏糖醇（DTT）。在隔膜当天新增DTT。 </li><li>缓冲液2:10mM HEPES，pH 7.4; 1mM EGTA; 1mM MgCl 2 ;和1mM DTT。在隔膜当天新增DTT。 </li></ol></li><li>从培养箱中取出转染的细胞（步骤1.4），吸出培养基，并用5 mL冰冷的磷酸盐缓冲盐水（PBS）冲洗细胞。吸出PBS和多余的液体碟子。 </li><li>向细胞中加入600μLBuffer 1。使用细胞刮刀，将细胞从板表面移开，并将细胞悬液移液至1.6 mL微量离心管中。 </li><li>立即在液氮中快速冷冻微量离心管。样品冷冻后，将溶解产物在冰上解冻，或将其置于-80°C下长时间储存​​。 </li><li>对所有细胞板重复步骤2.3和2.4。 </li><li> 一旦裂解物解冻，使用单脉冲微管匀浆器打开细胞。将均化器脉冲3-5次，持续10秒，将管放回冰上30秒钟之间。 <ol><li>收集20μL作为全细胞级分。 </li></ol></li><li>在1,000×g和4℃下将剩余样品离心10分钟。收集上清液并置于新的1.6 mL微量离心管中。 </li><li> 将沉淀物重悬于100μL缓冲液1中，并用研杵再造。脉冲同性恋生成器3-5次5秒钟，将管放回冰上30秒钟之间。 <ol><li>在1,000 xg和4°C下将样品再次离心10分钟。将上清液与步骤2.7的上清液合并。 注意:剩余的颗粒含有核和膜蛋白。 </li></ol></li><li>在11,000 xg和4℃下将合并的上清液离心20分钟。将上清液（细胞溶质级分）分离成新的1.6 mL微量离心管。 </li><li>将沉淀物重悬于200μL的缓冲液2中。将沉淀物均质化3-5次。在21,100 xg和4℃下将样品离心20分钟。吸出上清液。将含有粗膜级分的沉淀重悬于50μL缓冲液2中。 </li><li>立即进行GTPγS结合实验（第3部分）或在液氮中快速冷冻并储存在-80℃。 </li></ol> [ 35 S]GTPγS结合</p&gt; 注意:处理[ 35 S]GTPγS时和使用[ 35 S]GTPγS结合实验时，请使用标准放射化学安全性协议。随时戴防护手套和实验服。检查包装材料是否有泄漏或裂缝。根据制度方案处理废物和多余的试剂。 <ol><li>通过混合50mM HEPES，pH 7.4，制备不完全结合缓冲液（IBB）; 5mM MgCl 2; 100mM NaCl;和1mM乙二胺四乙酸（EDTA）。 </li><li>在10mM Tris-HCl，pH7.6和10mM DTT中稀释储备物[35S]GTPγS至50nM。制备500μL等分试样并将其储存在-80°C。在开始实验之前立即使用新鲜解冻的等分试样。在冰上解冻等分试样。 </li><li>通过将1mgGTPγS粉末溶解在177.62μL纯水中以制备10mM的原料浓度来制备非放射性标记的GTPγS。制作10-μL等分试样并存放-20°C。 </li><li>通过补充IBB来制备完整的结合缓冲液（CBB），包括终浓度为1mM DTT，0.1％（wt / vol）牛血清白蛋白（BSA），10μMGDP和0.1nM [35S]GTPγS。 注意:CBB现在包含放射性标记的GTP。在使用放射性溶液时采取适当的安全预防措施。 </li><li>在冰上从步骤2.11中解冻膜级分。 </li><li> 通过使用分光光度计在595nm的Bradford蛋白质测定来定量膜级分的蛋白质浓度。 <ol><li>用缓冲液2制备稀释系列的BSA蛋白标准品，终浓度为0,25,500,750,1,500,2,500和5,000μg/ mL。 </li><li>在比色杯中加入2μL每个标准品或膜1 mL的Bradford试剂。通过涡旋充分混合。 </li><li>使用0μg/ mL蛋白质的比色杯将分光光度计调整到595 nm波长，空白。 </li><li>等待5分钟，r每个标准和每个样品在595 nm波长。 </li><li>绘制标准品的吸光度与浓度。使用Beer-Lambert定律（A =εcl，其中ε=消光系数，l =比色皿长度，c =浓度），计算膜样品的浓度。 </li></ol></li><li>在IBB中将膜级分稀释至浓度为100μg蛋白/ mL。 注意:一个10厘米的HEK293细胞皿通常产生1和3毫克蛋白质/毫升之间。 </li><li> 在单个1.6 mL微量离心管中制备以下实验条件:配体稀释系列，测量基础GTP结合的基础活性条件和非特异性结合条件以测量非特异性GTP结合。 <ol><li>对于配体稀释系列，在最终体积为100μL的CBB中制备目标配体的稀释系列。将配体系列制备成2倍所需的最终浓度附件。空间配体浓度以充分覆盖一系列响应。 <ol><li>为了测定DAMGO对通过MOR1的GTP结合的影响，制备CBB中2mM，200μM，20μM，2μM，200nM，20nM，2nM，200pM，20pM和2pM的DAMGO稀释液体积:100μL）。 注意:例如，如果感兴趣的配体具有10μM的预期EC 50值，则制备200nM，2μM，20μM，200μM和2mM的配体稀释液。这五个条件涵盖了广泛的浓度，是测定所需浓度的2倍。 </li></ol></li><li>对于基底结合，只需准备100μLCBB的管。 </li><li>对于非特异性结合，制备配有99μLCBB的管，补充有1μL的2mM非放射性标记的GTPγS。 </li></ol></li><li>向每个实验条件中加入100μL稀释的膜溶液（步骤3.7）。 </li><li>孵化膜与各种实验在温热混合器或轨道振荡器中，在25℃下在1.6mL微量离心管中进行30分钟的条件。 </li></ol>膜过滤<ol><li>通过组合50mM Tris-HCl，pH 7.4制备洗涤缓冲液; 5mM MgCl 2 ;和50mM NaCl的蒸馏水中。 </li><li>将玻璃纤维过滤器预先放在水中10分钟。 注意:过滤器的孔径为1μm，直径为2.1cm，厚度为675μm。 </li><li>从温热混合器或轨道振动筛中取出样品（步骤3.10）。将样品短暂脉冲旋转5秒以收集管底部的每个样品。 </li><li>取下真空过滤装置的盖子。将预浸过滤器放置在设备的真空端口上。重新固定设备盖以形成真空密封。打开真空</li><li>将每个200μL实验条件的195μL吸管到过滤器上，以尽量减少吸附到管壁和/或移液管的误差婷。 </li><li>用1 mL冰冷的洗涤缓冲液清洗过滤器三次。 </li></ol>液体闪烁计数<ol><li>将5 mL闪烁计数小瓶放入计数架。向每个小瓶中加入5 mL闪烁液。 </li><li>关闭真空（步骤4.4）。取下真空过滤装置的盖子。使用镊子从过滤装置的真空端口中取出过滤器，并将每个过滤器放入单独的5 mL闪烁瓶中。 </li><li>用195μLCBB准备一个样品瓶，以确定最大信号。 </li><li>安全地盖住每个小瓶。在25°C的轨道振荡器上孵育小瓶10分钟。 </li><li>打开闪烁计数器。使用标准相关的闪烁程序对闪烁计数器进行编程，以测量每个样品35分钟同位素发射5分钟。 </li><li>按"开始"进行计数。 </li><li>计数完成后，处理计数的小瓶作为危险废物。 </li></ol>数据分析<ol><li> 将数据输入统计和分析软件。 <ol><li>从每个其他测量中减去非特异性结合以确定具体的结合。 注意:通过用非放射性标记的GTPγS孵育的样品测定非特异性结合程度（步骤3.8.3）。 </li><li>打开统计和分析软件并选择一个XY数据集条目。 </li><li>在[ 35 S]GTPγS结合实验中输入特定的结合数据到统计和分析软件中的数据表中。在"X"列中输入摩尔浓度的激动剂浓度。输入关联的35 S计数为"Y"值。 </li></ol></li><li> 转换数据。 <ol><li>通过单击"分析"将X值转换为各自的对数。从内置选择"变换"n分析。 </li><li>在"变换"对话框中，选择"使用变换X值"，然后选择"X = log（X）"功能。选择创建一个新的结果图。 </li><li>点击"确定"。 </li></ol></li><li> 归一化Y值。 <ol><li>显示变换结果后，单击"分析"。从内置分析中选择"规范化"。 </li><li>在"规范化"对话框中，选择单选按钮将0％定义为"每个数据集中的最小值"，100％作为"每个数据集中的最大值"。选择框以显示结果百分比。选择框以创建结果的新图形。 </li><li>点击"确定"。 </li></ol></li><li> 将非线性回归曲线拟合到绘制的数据。进行非线性回归分析。 <ol><li>显示标准化结果后，单击"分析"。从内置分析，选择"非线性回归（曲线拟合）"。 </li><li>如果调查激动剂，请从对话框中选择"log（激动剂）vs.归一化反应 - 可变斜率"。 </li><li>如果调查拮抗剂，则从对话框中选择对数（拮抗剂）与归一化反应 - 变量斜率。 </li><li>点击"确定"。 </li></ol></li><li>查看图表和结果，以确保合适和数据合理协议。确保回归与绘制数据的一般模式相匹配，并且残差不会大到使得剂量 - 反应曲线平坦。 注意:软件将总结非线性回归的结果，详细介绍引起半最大响应（EC 50 ），希尔系数（ H H ）和半最大抑制浓度（IC 50 ）的浓度。 </li><li> 衍生激动剂/拮抗剂参数效力。 <ol><li>推导出激动剂平衡解离常数（K b ）来自以下实验16,17,18 <ol><li>评估激动剂与固定浓度拮抗剂竞争的剂量反应的变化。这里EC 50 &#39;= EC 50 （1 + [拮抗剂] /拮抗剂Kb ），其中EC 50 &#39;是右移EC 50 。 </li><li>评估[ 35 S]GTPγS结合不同浓度的拮抗剂与固定浓度的激动剂竞争。这里，K b = IC 50 /（2 +（[激动剂] /激动剂EC 50 ） n ） 1 / n -1，其中n是激动剂的Hill系数。 </li></ol></li></ol></li><li>根据数据的变异性，对每个配体重复实验（步骤3.8-5.6）约3-5次，并使用统计和分析软件对结果进行平均是。 </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Assessment+of+M1+Muscarinic+Acetylcholine+Receptor-Gq/11+Protein+Coupling+in+Membranes+Prepared+from+Postmortem+Human+Brain+Tissue.">Pharmacological Assessment of M1 Muscarinic Acetylcholine Receptor-Gq/11 Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue.</a> J. Pharm. Exp. Ther. 325 (3), 869-874 (2008).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles:+extending+the+utility+of+[35S]GTP+gamma+S+binding+assays.">Principles: extending the utility of [35S]GTP gamma S binding assays.</a> Trends. Pharmacol. Sci. 24 (2), 87-90 (2003).</li><li>Harrison, C., Traynor, 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=The+[35S]GTPgammaS+binding+assay:+approaches+and+applications+in+pharmacology.">The [35S]GTPgammaS binding assay: approaches and applications in pharmacology.</a> Life Sci. 74 (4), 489-508 (2003).</li><li>Traynor, J. R., Nahorski, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+by+mu-opioid+agonists+of+guanosine-5'-O-(3-[35S]thio)triphosphate+binding+to+membranes+from+human+neuroblastoma+SH-SY5Y+cells.">Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells.</a> Mol. Pharmacol. 47 (4), 848-854 (1995).</li><li>Sittampalam, G. 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=GTP&#947;S+Binding+Assays.">GTP&#947;S Binding Assays.</a> Assay Guidance Manual. , (2004).</li><li>Strange, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+GTP&#947;S+([35S]GTP&#947;S+and+Eu-GTP&#947;S)+binding+assay+for+analysis+of+ligand+potency+and+efficacy+at+G+protein-coupled+receptors.">Use of the GTP&#947;S ([35S]GTP&#947;S and Eu-GTP&#947;S) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors.</a> Br. J. Pharmacol. 161 (6), 1238-1249 (2010).</li></ol>

作者宣称没有竞争的兴趣。

本方案描述了两种单独但互补的方法:将细胞和组织分成广泛但不同的隔室的简单方法，以及通过测量[ 35 S]GTPγS结合来研究GPCR信号的方法。 有效的细胞分馏具有广泛的应用范围，从蛋白质的提取和富集到蛋白质亚细胞定位的评估，到受体药理学的研究。虽然存在分选细胞和组织的替代方法，但是这里提出的方案比较便宜，更快，更简单。然而，与更成熟的方法不同，该方法不能将质膜相关蛋白与其他膜隔室（例如内体或ER）干净地分离。还必须指出，虽然上述方案使用瞬时过表达系统来产生含有GPCR的细胞膜兴趣，该协议与稳定的过表达系统以及动物或人体组织相容19 。 样品制备对于最大化馏分产率和纯度至关重要。确保完全均匀化并使离心步骤之间的转换时间最小化是特别重要的。使用颗粒匀浆器/杵完全破坏细胞膜显着增加了最终细胞部分中的粗制膜产量。如果膜产量太低，则两个最简单的解决方案是将初始细胞培养物放大和/或使细胞沉淀物均匀化几次。如果各个级分显示出高度的杂质，则可减少离心和分离之间的转换时间。离心后不要让样品静置，因为蛋白质可能扩散到样品中并降低成分的纯度。 基于过滤的[ 35 S]GTPγS结合是使用相关受体测量G蛋白活化的快速和定量的方法。测量GTP结合允许对监测下游过程时通常可能的GPCR活性进行更定量的测量。然而，这里概述的方法有两个关键的限制。最重要的是，基于过滤的[ 35 S]GTPγS定量与所有Gα同种型20,21不同 。一般来说，这种方法仅限于像MOR1 22这样的G i / o耦合GPCR。 G s - 和G q-偶联受体往往不太敏感。这可能是由于G s / G q的丰度较低及其相对较慢的核苷酸交换率的组合，使得难以区分背景上的真实[ 35 S]GTPγS结合。这个限制已经被解决了<html> e使用G蛋白抗体捕获技术20 。一些团体发现，消除GDP从反应中可以改善G s或G q耦合的受体23的信噪比。第二个限制是对亲脂性激动剂或表面活性剂的膜敏感性。 [ 35 S]GTPγS测定需要功能性受体-G-蛋白复合物，并且向反应体系中加入亲脂性分子可能破坏膜或这些复合物的结构。如果这是一个潜在的问题，可能有利的是测试目的试剂是否已经在已经很好表征的系统中结合（例如在DAMGO介导的MOR1刺激中）[ 35 S]GTPγS结合。考虑优化的其他条件包括结合缓冲液pH，Mg 2+和NaCl的浓度 ，蛋白质浓度和孵育时间23 ，ef"&gt; 24。 该方案集中在作为MOR1的良好表征的GPCR，MOR1和良好表征的药物DAMGO（激动剂）和纳洛酮（拮抗剂）。然而，这种技术的优点之一是它可用于表征未知配体作为激动剂，拮抗剂或反向激动剂，这取决于配体如何调节GTP结合。在设计实验时，使用一系列浓度的目标配体至关重要，并注意结果范围:激动剂会导致GTP结合增加，而反向激动剂会导致GTP结合的降低基线和拮抗剂在基线GTP结合时分离加入时几乎没有影响。 总之，该方案描述了一种用于进行亚细胞分离，收集粗膜制剂和通过测量[ 35 S] G检测GPCR活化的技术TPγS结合。这些技术可以容易地适应于各种细胞培养和组织模型，以研究最重要的受体家族之一的药理学。

G蛋白偶联受体（GPCR）是一系列大量的细胞表面受体，负责一系列生理过程，包括镇痛，嗅觉和行为1 。 GPCR通过检测特定的外部信号并随后刺激细胞内信号传导而起作用。因此，它们标志着电池的外部和内部环境之间的关键连接。由于GPCR在生物学中的关键作用，它们已经成为基础研究和药物发现的主要目标2,3 。 与其他结合离散配体的受体家族不同，GPCR可以结合非常不同类型的分子。虽然一个GPCR可能与肽相互作用，但另一个可能会感测到光子，小分子或离子1,4 。虽然他们的配体是多样的，GPCR在他们的总体架构师是统一的ure和功能。单个GPCR由七个具有细胞外氨基末端和细胞内羧基末端的α螺旋跨膜蛋白5,6组成 。 GPCR与由α，β和γ亚基组成的细胞内G-蛋白 - 异源三聚蛋白复合物偶联 - 介导多种信号通路7 。 Gα亚基是鸟嘌呤核苷酸结合蛋白，当与鸟苷二磷酸（GDP）结合时是无活性的，当与鸟苷三磷酸（GTP）结合时，其活性为8,9 。当GPCR结合其配体时，它们经历构象变化，允许Gα从Gβγ解离，从而允许Gα与GTP 7交换GDP。受体本身在其羧基末端被各种丝氨酸/苏氨酸磷酸化内源性激酶10,11并且内化以减弱受体信号传导12,13,14 。同时，激活的Gα单体和Gβγ二聚体进行激活不同的信号通路7 。每个G蛋白亚基有几种同种型，每种同种型都针对特定的下游途径和二级信使系统。主要的Gα同种型包括G s ，G q ，G i / o和G 12-13 。通常，单个GPCR与特定的Gα同种型相关联，从而将外部刺激与特异性细胞反应1相联系 。 表征GPCR-配体相互作用对于了解受体的生物学至关重要。由于GDP / GTP交易是最早的一天遵循配体结合的nts，监测GTP结合可以测量GPCR活化或抑制。在GPCR信号传导中测定更多的下游事件通常不是定量的或化学计量的，可能不区分完全激动剂与部分激动剂，并且可能需要昂贵的试剂。此外，增加的GTP与Gα蛋白的结合是GPCR激活后几乎是普遍的事件，这意味着测量GTP结合是广泛适用于监测大多数GPCR的活性的测定。测量GTP结合是一种简单而快速的方法来监测过表达感兴趣受体或天然组织的细胞中的GPCR信号。本方案详细描述了使用原型GPCR（μ-阿片受体（MOR1））的功能性GTP结合测定来定量测定激动剂和拮抗剂对GPCR信号传导的活性。 该方案首先概述了如何从过表达MOR1的细胞中分离粗膜。注意到该方案不限于过表达系统，并且可以应用于许多来源的膜，包括表达多种受体和G蛋白的天然组织或制剂。然后该方案详细说明如何响应不同浓度的[D-Ala，N-MePhe，Gly-ol] - 脑啡肽（DAMGO）或纳洛酮（MOR1激动剂和拮抗剂）测量放射性GTP类似物与这些膜的结合，分别。 GTP类似物[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）是不可水解的。该性质是至关重要的，因为Gα亚基表现出内在的GTPase活性7 ，并且可消除可水解的GTP放射化学上的标记的γ磷酸盐。然后将膜捕获到玻璃纤维过滤器上并洗涤，之后通过液体闪烁计数定量放射性标记的GTP。可以推导出多个药理学参数包括受体 - 配体相互作用，包括激动剂的半最大反应（EC 50 ）和希尔系数（n H ）以及拮抗剂16,17的半最大抑制浓度（IC 50 ）和平衡解离常数（K b ） ， 18 。

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DMEM, high glucose, pyruvate, no glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">10313021</td>
			<td style="width:264px;">Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">L-glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">25030081</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Penicillin-Streptomycin</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">15140122</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">31985070</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Fetal Bovine Serum</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Cell culture 10-cm plate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Lipofectamine 3000 reagent</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L3000-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">1.6 mL microcentrifuge tubes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">1615-5500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Tris(hydroxymethyl)aminomethane (Trizma base)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sucrose</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">2900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">DO632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:463px;">Magnesium chloride (MgCl2)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1028-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pellet pestles motor</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z359971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pestles</td>
			<td style="width:189px;">Bel Art</td>
			<td style="width:119px;">F19923-0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bovine serum albumin (BSA)</td>
			<td style="width:189px;">Affymetrix</td>
			<td align="right" style="width:119px;">10857</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[35S]guanosine-5&#8217;-O-(3-thio)triphosphate ([35S]GTP&#947;S)&#160;</td>
			<td style="width:189px;">Perkin Elmer</td>
			<td style="width:119px;">NEG030H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">non-radiolabeled guanosine-5&#8217;-O-(3-thio)triphosphate (GTP&#947;S)&#160;</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">89378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">guanosine diphosphate (GDP)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">51060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bradford reagent</td>
			<td style="width:189px;">Bio-Rad</td>
			<td align="right" style="width:119px;">5000006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">UV/VIS spectrophotometer</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">DU640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">spectrophotometer cuvettes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">9090-0460</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">orbital shaker</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">2314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">thermomixer</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">535027903</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">glass fiber filters&#160;</td>
			<td style="width:189px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">1821-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum filtration apparatus</td>
			<td style="width:189px;">Millipore Corporation</td>
			<td style="width:119px;">XX2702550</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">desktop microcentrifuge</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">65717</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Scintillation counter</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">LS6500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation fluid&#160;</td>
			<td style="width:189px;">Ecoscint A</td>
			<td style="width:119px;">LS-273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation counter vials</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation vial lids</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Prism 6</td>
			<td style="width:189px;">GraphPad Software</td>
			<td style="width:119px;">PRISM 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ATP1A1 antibody</td>
			<td style="width:189px;">Developmental Studies Hybridoma</td>
			<td style="width:119px;">a6F</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">GAPDH antibody</td>
			<td style="width:189px;">EMD Millipore</td>
			<td style="width:119px;">CB1001</td>
			<td>1:5000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">H2B antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">2934S</td>
			<td>1:2500 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">PDI antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">3501S</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">HA antibody</td>
			<td style="width:189px;">Roche</td>
			<td align="right" style="width:119px;">11867423001</td>
			<td>1:2000 in 3% BSA</td>
		</tr>
	</tbody>
</table>

measuring g-protein-coupled receptor signaling via radio-labeled gtp binding

G-蛋白偶联受体 （GPCR）是一系列跨膜受体，在正常细胞生理学中起关键作用，构成多种适应症的主要药理学靶点，包括镇痛，血压调节和精神病治疗。在配体结合后，GPCRs通过刺激三磷酸鸟苷（GTP）的掺入来催化胞内G蛋白的活化。活化的G蛋白刺激引发细胞反应的信号通路。可以通过测量GTP，[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的放射性标记和不可水解形式掺入G蛋白来监测GPCR信号。与评估更多下游信号传导过程的其他方法不同，[ 35 S]GTPγS结合测量GPCR信号中的近端事件，并且重要的是可以区分激动剂ts，拮抗剂和反向激动剂。本方案概述了使用原型GPCR（μ-阿片样物质受体（MOR1））的粗膜制剂研究GPCR信号的敏感和具体方法。虽然存在分离细胞和组织的替代方法，但许多成本过高，繁琐和/或需要非标准的实验室设备。本方法提供了丰富功能性粗膜的简单方法。分离MOR1后，测定其激动剂[D-Ala，N-MePhe，Gly-ol] - 脑啡肽（DAMGO）和拮抗剂纳洛酮的各种药理学性质。

细胞分离可用于从细胞溶质和核蛋白中分离和富集膜相关蛋白。 图1是Western印迹，证实在亚细胞分馏过程中可以收集的三种主要级分的含量。具体来说， 图1显示了组蛋白H2B和甘油醛-3-磷酸脱氢酶（GAPDH），核蛋白和胞质蛋白质的分离干净地分离膜蛋白（ 即 Na + / K + ATP酶，蛋白质二硫键异构酶（PDI）和HA-MOR1）分别。另外， 图1显示了作为分级结果的蛋白质（泳道1与泳道4相比）在其各自的亚细胞级分中的富集。代表性的Ponceau染色显示在每个级分中相等的蛋白质载量。重要的是要注意这个分解onation协议不区分不同的细胞膜。 PDI通常定位于内质网（ER），而Na + / K + ATP酶主要位于质膜上。然而，两种蛋白质都存在于最终的粗制膜部分（ 图1 ，泳道4）中。另外，尽管该协议强力地将核蛋白与细胞溶质和最终膜级分（ 图1 ，泳道2-4）分离，但富含核蛋白的级分含有一些膜蛋白（ 图1 ，泳道2），可能来自ER。 可以通过GTP结合实验（ 表1 ）得出多重药理学参数以表征GPCR-配体相互作用。例如，激动剂的半最大反应（EC 50 ）和希尔系数（n H ）可以通过监测GTP结合对不同剂量的激动剂的反应。 图3A显示DAMGO处理后剂量反应性GTP与MOR1的结合。当数据适合四参数非线性回归时，拟合描述了受体 - 配体与185±23nM的EC 50和0.46±0.06的Hill系数的相互作用。 DAMGO治疗后观察到的浅GTP结合曲线表明DAMGO和MOR1之间的负相关性。该方法还可以鉴定和描述拮抗剂的药理学。 如图3A和B所示，纳络酮是MOR1拮抗剂。通过改变与固定浓度的DAMGO竞争的纳洛酮浓度来确定纳洛酮的激动剂效力（K b ），97±20nM（ 图3A ）。纳洛酮显示出0.88±0.06的Hill系数，表明纳洛酮与MOR1之间的独立结合。如果交流配体是未知的，该测定可以区分激动剂，拮抗剂和反向激动剂。如果配体是激动剂，则在DAMGO应用后， 如图3A所示 ，GTP结合将会增加。如果配体是反向激动剂，则GTP相对于基础结合的结合将减少。如果配体是拮抗剂，则仅对配体进行治疗将不起作用。如果与激动剂同时使用，拮抗剂将抑制激动剂刺激GTP结合的能力。 图3A说明纳洛酮对激动剂DAMGO的拮抗剂活性。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55561/55561fig1.jpg" /> 图1:细胞分离分离膜相关的，核的和胞质蛋白。 （ 上 ）第1行代表蛋白质存在于整个细胞中。泳道2包含在第一离心步骤期间分离的核和膜相关蛋白。泳道3是在11,000×g离心20分钟后分离的胞质级分。泳道4含有适用于[ 35 S]GTPγS结合实验的粗膜级分。 （ 底部 ）Western膜的Ponceau染色显示每个细胞部分的蛋白质负载。以下抗体用于免疫印迹:小鼠抗Na + / K + -ATPase（1:1,000），小鼠抗GAPDH（1:5000），小鼠抗H2B（1:2,500），兔抗PDI（1 :1,000）和大鼠抗HA（1:2000）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55561/55561fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55561/55561fig2.jpg" /> 图2流程图概述了分级和[ 35 S]GTPγS结合方法。首先，转染细胞以表达感兴趣的GPCR。 48小时后，收获细胞并分离细胞以分离受体（红色）和相关G蛋白（绿色= Gα，紫色= Gβ和橙色= Gγ）。为了进行GTP结合实验，加入[ 35 S]GTPγS，并用感兴趣的配体孵育膜。为了测量G蛋白活性，将膜结合到过滤器上以洗去任何未结合的放射化学物质，然后通过液体闪烁计数定量结合的[ 35 S]GTPγS。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55561/55561fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55561/55561fig3.jpg" /> 图3:激进a在[ 35 S]GTPγS结合定义的μ-阿片样受体拮抗剂。 （ A ）与2.5μMDAMGO（绿色）竞争时，单独使用DAMGO（蓝色）或纳洛酮的[ 35 S]GTPγS剂量 - 反应曲线。 [ 35 S]GTPγS结合标准化为每个实验中的最大刺激，并以百分比表示。所示的点是三次测定的平均值，并表示为平均值±SEM（ B ）DAMGO刺激的[ 35 S]GTPγS的拮抗作用 纳洛酮结合。 [ 35 S]GTPγS 在纳洛酮（100μM）与纳洛酮（100μM）竞争中，单独加入纳洛酮（100μM），单独使用DAMGO（10μM）或DAMGO（10μM）后，定量结合。结果表示为三次独立实验的平均值±SEM。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55561/55561fig3large.jpg" target="_blank">请点击他</a>重新查看这个数字的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 配体 </td><td> EC 50或IC 50 （nM） </td><td> n H </td><td> K b （nM） </td></tr><tr><td> DAMGO </td><td> 185±23 </td><td> 0.46±0.06 </td><td></td></tr><tr><td>纳洛酮</td><td> 420±87 </td><td> 0.88±0.06 </td><td> 97±20 </td></tr></tbody></table> 表1:μ-阿片样物质受体的DAMGO和纳洛酮活性的药理学参数。 DAMGO的半最大反应（EC 50 ）和Hill系数（n H ）来自响应于不同剂量DAMGO的[35S]GTPγS结合。半最大抑制浓度（IC 50 ）和纳洛酮的平衡解离常数（K b ）由纳洛酮与2.5μMDAMGO在[ 35 S]GTPγS结合上的竞争的影响而确定。结果表示为三次独立实验的平均值±SEM。

Watch this Scientific Journal Video about Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding at JoVE.com

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

三磷酸鸟苷（GTP）结合是G蛋白偶联受体（GPCR）激活中最早的事件之一。该方案描述了如何通过监测放射性标记的GTP类似物[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的结合在药理学上表征特异性GPCR-配体相互作用对感兴趣配体的反应。

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

measuring-g-protein-coupled-receptor-signaling-via-radio-labeled-gtp

Nanyang Technological University

Research

Education

JoVE Journal

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Biochemistry

Cell Biology

Pharmacology

Unit 1

Pharmacodynamics

Signaling Networks of G Protein-coupled Receptors

SCIENTISTS IN ACTION

16.2K 次观看.  Johns Hopkins University School of Medicine. 三磷酸鸟苷（GTP）结合是G蛋白偶联受体（GPCR）激活中最早的事件之一。该方案描述了如何通过监测放射性标记的GTP类似物[ 35 S]鸟苷-5'-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的结合在药理学上表征特异性GPCR-配体相互作用对感兴趣配体的反应。 

视频: Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

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

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

通过共沉淀测量蛋白与F-肌动蛋白的结合

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

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...