Name: an elisa based binding and competition method to rapidly determine ligand-receptor interactions
Uploaded: 2016-03-14T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions at JoVE.com

We thank Prof. J. Stelling (Department of Biosystems Science and Engineering, ETH Zurich and Swiss Institute for Bioinformatics, Basel, Switzerland) for his critical review of the manuscript.

1.试剂准备<ol><li>以制备碳酸包被缓冲液，溶解0.36克Na 2 CO 3和0.84克的NaHCO 3在100毫升蒸馏水;无菌过滤器通过使用真空从动0.22μm的聚醚砜（PES）膜过滤器，并存储在RT之前使用的缓冲区。 </li><li>通过在磷酸盐中加入0.05％体积/体积的吐温20制备洗涤溶液缓冲盐水（PBS）。 </li><li>通过在100毫升PBS中5克BSA（≥98％），并存储溶解在4℃下制备的5％牛血清白蛋白（BSA）的PBS溶液（封闭溶液）。 </li><li>重组受体，配体和阻止肽<ol><li> Reconstitutethe重组人白介素受体α亚基（IL28RA），并根据制造商的说明，并储存于-80℃的重组人干扰素（IFNL1-3）的His标记的配体。 Synthetize阻断肽和如先前所述4中使用。使用PBS中制备不同浓度的配体的秒和在测定中使用的肽。 </li></ol></li><li>为了制备第一抗体，稀释在PBS中的0.1％BSA的6倍他小鼠单克隆抗体以1:1000稀释。为了制备第二抗体，稀释辣根过氧化物酶（HRP）的PBS缀合的山羊抗小鼠IgG（H + L）含有1 0.1％BSA的1:10,000稀释。 </li><li>通过根据制造商的说明混合试剂A和B制备TMB溶液。 </li><li>通过在RT加入在蒸馏水和商店5 N硫酸（H 2 SO 4）准备终止溶液。 </li></ol> 2.酶联免疫测定（ELISA） 注:直接配体-受体相互作用的ELISA（直接LRA， 图1）可以被用于测量受体-配体的解离常数（K D），作为受体-配体结合亲和力的量度。竞争配体-受体相互作用的ELISA（竞争LRA， 图2）的所有流入的肽（和其他阻断化合物），其作用与配体和受体之间的相互作用来干扰的筛选。先前公布5的基本协议进一步优化。 注意:在这两种ELISA方法使用多道移液器用于添加解决方案，以在每一个步骤96孔板的孔中。在溶液澄清或洗涤步骤，直接扔出去的解决方案整合到水槽。 <ol><li>直接配体 - 受体相互作用测定（直接LRA） 注意:对于该工作流的示图（ 见图1）。 <ol><li>涂层板的重组受体<ol><li>稀释在碳酸盐缓冲液的重组受体至100毫微克/微升的最终浓度。通过使用多道移液器以及移液100微升到每个具有固定的受体浓度（100毫微克/微升）96孔微量滴定板的涂层的孔中。排除板外墙面，以免以及边缘神器。覆盖有盖板，孵育的板在4°CO / N。 </li></ol></li><li>阻塞和配体的加入<ol><li>第二天，通过倾斜靠在水槽板除去涂层溶液和3次洗涤液（PBS + 0.05％体积/体积的吐温20）洗涤该板。 </li><li>用200微升的5％BSA溶液到每个用多道移液器井框在涂板的游离受体结合位点和孵育所述板在室温2小时。 </li><li>弃去封闭溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li><li>制备重组His-标记的配体在不同浓度（ 例如，8微克/毫升，4微克/毫升，2微克/毫升，1微克/毫升，0.5微克/毫升，0.25微克/毫升，0.125微克/毫升，0.063微克/毫升，0.031微克/毫升，0.0微克/毫升）的PBS。仅添加PBS的空白孔。 </li><li>加入100微升每配体浓度向孔中一式两份，并在室温孵育2小时的板允许受体 - 配体相互作用。 </li></ol></li><li>孵化与抗体<ol><li>与配体孵育后，3次洗涤平板用洗涤溶液。 </li><li>吸取100μl的初级抗His小鼠单克隆抗体溶液（1:1,000）的每个孔中。 </li><li>孵育在室温下2小时的板;温育后，弃去抗体溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li><li>添加100微升HRP偶联的山羊抗小鼠IgG二抗溶液（1:10,000）到每个孔中。将培养板在室温下45分钟。 </li><li>弃去抗体溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li></ol></li><li>基材与发展加成<ol><li>使TMB底物溶液到室温，并在1制备TMB底物溶液A和B:1的比例。添加新鲜制备的衬底以每孔100μl，并保持在室温的板15-30分钟。经过充分的山坳<html>或开发加入50微升一站式解决方案。 </li></ol></li><li>读板和数据分析 注意:所述的协议是基于所测量的信号从特异性结合升起的假定。它可能是估算非特异性结合到该信号的贡献必要的，但是这是出于这个协议的范围。 <ol><li>直接读取450nm处的吸光度（光密度，OD值）。 </li><li>减去从测量OD值的背景信号和正常化它们。变换的所有值的配位体浓度为对数标度的（基座10，日志10）。 </li><li>积归一化和背景校正OD值（Y轴，对应于被占领受体结合位点的分数）抗配体浓度的对数（X轴，对数10的范围）。 </li><li>为了估计在K D值，拟合数据到Hill方程的形式如下: tp_upload / 53575 / 53575eq1.jpg"/&gt; 注意:这里Y代表占据受体结合位点和Y 最大最大结合的部分; [L]表示游离配位体和希尔系数的浓度。如果只有一个用于配体结合位点，希尔系数为n = 1，对于具有多于一个的配位体结合位点的系统中，结合展品正协同如果n&gt; 1，负协同如果n &lt;1和没有协同如果n = 1，微观解离常数被称为并对应于半数最大有效浓度EC 50 6。表观解离常数为K D =（K D）ñ。在最简单的，其中n = 1的情况下，解离常数对应于该受体结合位点的一半被占用和KÐ= K D中的配体浓度。这个模型假定质量作用在平衡条件下的结合，以及，只有一小部分加入的配位体与受体结合， 即，[L] &gt;&gt; [RL]。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/53575/53575fig1.jpg" /> 图1. 直接配体-受体相互作用测定（直接LRA）。直接LRA一步一步的协议。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53575/53575fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>竞争配体 - 受体相互作用测定（竞争LRA） 注意:对于该工作流的示意图参见图2中的竞争LRA程序遵循作为直接LRA除了在配体的重要变化相同的步骤（涂布板，抗体孵育，板发展）和肽加成步骤。合适的阴性对照是用于该测定是必不可少的。在以前的筛选研究&lt;SUP&gt;如图4所示，加扰的封闭肽没有表现出拮抗作用。 <ol><li>阻塞 - 配体和阻止肽加成<ol><li>第二天，除去涂层溶液和洗涤所述板（见2.1.2.1）。 </li><li>通过加入200微升的5％BSA溶液，以每孔阻挡涂板，孵育该板在RT 2小时。 </li><li>在固定的浓度（2×20毫微克/毫升）的PBS制备重组His-标记的配体（IFNL1-3）。 </li><li>制备具有不同浓度范围从10nM至100μM的在PBS中，以保证一个剂量-反应曲线的阻塞肽（参照表3）。 注:此启用阻断肽的IC 50值的连续测定。在对照孔，只加固定的配位体浓度没有肽获得最大（100％）的结合。在一片空白，只添加PBS没有配体或肽。 </li><li>加的配位体的50微升（IFNL1-3）和50微升各PE的ptide集中在重复的井。 </li><li>将培养板在室温2小时。 </li></ol></li><li>读板和数据分析 注意:所述的协议是基于所测量的信号从特异性结合升起的假定。它可能是估算非特异性结合到该信号的贡献必要的，但是这是出于这个协议的范围。 <ol><li>直接读取450nm处的吸光度（光密度，OD值）。 </li><li>减去从测量OD值的背景信号和正常化它们。变换的所有值的肽浓度对数标度的（基座10，日志10）。 </li><li>积归一化和背景校正OD值（Y轴，对应于被占领受体结合位点的分数）抗配体浓度的对数（X轴，对数10的范围）。 </li><li>来估计IC 50值，与数据拟合到以下公式: <img alt="公式（2）" src="/files/ftp_upload/53575/53575eq2.jpg" /> 注:此处[P]为肽浓度和Hill斜率。 Hill斜率描述了剂量 - 反应曲线的陡度。将IC 50对应于被观察到的配体和受体之间的结合的50％抑制的抑制剂浓度。 </li></ol></li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53575/53575fig2.jpg" /> 图2.竞争配体-受体相互作用测定（竞争LRA）。竞争LRA一步一步的协议。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53575/53575fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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A.E. was supported by a research grants from the "SNSF Ambizione Score" program (PZ00P3_154709), "Forschungsfond, F&#246;rderung strategischer Projekte" University of Basel, Stiftungsinfektionskrankheiten Basel, and Bangeter Rhyner Stiftung. J.L. acknowledges support by an iPhD fellowship of the SystemsX.ch initiative in systems biology program (9th call).

ELISA是许多实验室标准和行之有效的方法。我们进一步修改和完善了先前公布的方法5,7。所表现出的一步一步的协议说明了如何能够以简单的方式被用于确定配体-受体相互作用的K个D取值。此外，阻挡肽，与配体 - 受体相互作用干扰的IC 50可被确定。 主要优点是快速安装，易于制备的试剂和熟悉的操作，因为大多数研究人员以前使用的ELISA协议。直接LRA协议是...

信号通路的全面了解需要有关配体 - 受体相互作用的详细知识。评估其特异性受体的特定配体的相互作用的大多数方法是昂贵的，耗时的，劳动密集型的，并且需要特定的设备和专门知识1。 本文介绍了两个快速和可靠的逐点的协议来调查的基础上的一种酶的配体 - 受体相互作用酶联免疫吸附测定（ELISA）:直接配体 - 受体相互作用测定（LRA）和竞争LRA。 ELISA是一个高度敏感的，具体的和随时可用的技术，几乎在每一个实验室常规使用。 ELISA可以执行并适于各种方式。所提出的协议对不同的λ干扰素（INFLs）和它们的受体之间的相互作用的调查进行了优化。 直接LRA允许一个quantificati关于配体 - 受体的相对于配体浓度结合和由此产生的结合曲线。使用适当的型号为配体-受体相互作用，可以将数据进一步分析以估计的解离常数（K D）。 在所提出的协议，通常使用希尔方程被施加到该配体 - 受体结合建模。尽管其它方法，例如表面等离子共振技术2,3-允许两个蛋白之间的结合亲和力的测定，这种技术常常是劳动密集型的，昂贵的，并且需要特殊的实验室设备。 竞争LRA能够抑制肽的筛选:在配体 - 受体结合相对于肽浓度定量。这产生描述的肽的抑制作用的剂量 - 反应曲线。数据可以被进一步分析以估计半数最大抑制浓度（IC 50 </子&gt;）阻断肽。 两者的ELISA协议是易于使用，并且可以适合于广泛的研究问题。任何形式的重组蛋白可以用于可靠地并快速确定相互作用的部件。此外，竞争LRA可用于通过使用阻断肽，其目的是为了模仿或配体或受体，以确定配体和受体的关键相互作用位点。如果封闭肽显示有效和特异性抑制，肽占据配体的关键相互作用位点（如肽模拟物的受体）或配体（如肽模拟物的配位体）。 第一个协议描述不同INFLs的在K D值的决心和其受体α亚基， 即使用直接LRA白细胞介素28受体（IL28RA）。接着，第二协议显示如何确定一个20个氨基酸长的肽的能力抑制INFL-IL28RA相互作用。肽被设计成在其受体结合位点与IFNLs竞争，从而使相互作用的分子的理解。此外，该肽可以用于阻止在体外实验IL28RA以确定对下游信号效应4的影响。

<table><tbody><tr><td>Nunc-Immunoplate (F96 Maxi sorp)</td><td>Thermo Scientific</td><td>442404</td><td>ELISA plate</td></tr><tr><td>Sodium carbonate (Na2CO3)</td><td>Merck</td><td>497-19-8</td><td>For ELISA plate coating buffer</td></tr><tr><td>Sodium hydrogen carbomnate(NaHCO3)</td><td>Merck</td><td>144-55-8</td><td>For ELISA plate coating buffer</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7030-100G</td><td>5% BSA in PBS for Blocking</td></tr><tr><td>rhIL-28R&amp;alpha;/IFN&amp;lambda;R1</td><td>R&amp;amp;D systems</td><td>5260-MR</td><td>Recombinant human interlukin-28 Receptor alpha</td></tr><tr><td>rhIL-29/IFN&amp;lambda;1</td><td>R&amp;amp;D systems</td><td>1598-IL/CF</td><td>Recombinant human interlukin-29/Carrier free/C-terminal 10-His tag</td></tr><tr><td>rhIL-28A/IFN&amp;lambda;2</td><td>R&amp;amp;D systems</td><td>1587-IL/CF</td><td>Recombinant human interlukin-28A/Carrier free/C-terminal 6-His tag</td></tr><tr><td>rhIL-28B/IFN&amp;lambda;3</td><td>R&amp;amp;D systems</td><td>5259-IL/CF</td><td>Recombinant human interlukin-28B/Carrier free/C-terminal 6-His tag</td></tr><tr><td>6x His Monoclonal antibody (Mouse)</td><td>Clontech</td><td>631212</td><td>Primary antiboy to capture His tagged Ligands</td></tr><tr><td>Goat anti-Mouse igG (H+L)</td><td>Jackson Immuno Research</td><td>115-035-166</td><td>Horseradish Peroxidase conjucated secondary antibody</td></tr><tr><td>BDoptEIA TMB reagent set</td><td>BD Biosciences</td><td>555214</td><td>ELISA - TMB substrate solution</td></tr><tr><td>Sulfuric acid (H2SO4)</td><td>Fulka</td><td>84720</td><td>5 N H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; (Enzyme reaction stop solution)</td></tr><tr><td>Synergy/H1 - Microplate reader</td><td>BioTeK</td><td></td><td>ELISA plate reader</td></tr></tbody></table>

an elisa based binding and competition method to rapidly determine ligand-receptor interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

使用直接LRA确定INFL1-3及其受体α亚基IL28RA之间的解离常数。结果示于图3:所占据的结合位点的小数值靠着相应的IFN浓度的对数作图。数据的Scatchard作图示于右下角。的结果表明，直接LRA产生一个结合曲线，其可以被进一步分析，以估计在K D值。在K D值，通过拟合数据至希尔方程（方程1）测定。 IFNL...

Watch this Scientific Journal Video about An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions at JoVE.com

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

an-elisa-based-binding-competition-method-to-rapidly-determine-ligand

Universidad San Sebastian

Research

JoVE Journal

Chemistry

19.0K 次观看.  University of Basel. The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions. 

视频: An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

科研

化学

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

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

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 ...

生物化学

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

神经科学

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

生物学

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

Source: Singh, R. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59635">Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins.</a> J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

基于 SELEX 的体外结合测定法，用于鉴定 RNA-蛋白质相互作用

Source: Singh, R. Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins. J. Vis. Exp. ( (2019).
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the ligand-binding site. The specificity of a protein&#8217;s ligand-binding site is determined by the arrangement of its amino acid chain which gives the area its shape and chemical reactivity. Hence, a ligand-binding site provides a complementary shape to its ligand and keeps the ligand in place via chemical interactions. These chemical interactions are often noncovalent; however, since these interactions are reversible and weak, many of these interactions need to occur simultaneously to hold the protein and the ligand together.
Research that elucidates interaction mechanisms at ligand binding sites generally involves in silico modeling and in vitro approaches. In silico modeling uses computers to compare previously known protein structures and evolutionary data to make predictions to determine the optimal binding shape and energy state of the protein-ligand complex. In vitro approaches compliment in silico predictions by providing evidence for ligand binding through binding and kinetic assays in the laboratory. Ligand binding research is important for understanding the functions of proteins and how they perform specific cellular processes in both healthy, as well as in diseased conditions. For instance, certain genetic conditions and cancers can alter the sequence of a protein, ultimately affecting its ability to bind a ligand. In addition, this research also allows scientists to design drugs with specific interactions and minimal side effects by targeting the ligand-binding site of an implicated protein.

Ligand Binding Sites and Protein-Ligand Interactions

配体结合位点

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their ...

教育

JoVE Core

分子生物学

Unit 1

Protein Function

关键术语与概念

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center;" />
where&#160; P and L are the unbound protein and ligand, respectively, and PL is the protein-ligand complex.&#160;
As the amount of bound ligand is also related to the rate of ligand binding, experiments can also determine Kb by examining the rates of protein-ligand association (kon) and dissociation (koff) using the following ratio:
<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;" />
Thus, two categories of&#160; binding assays are used to determine the equilibrium binding constant &#8211; those that measure equilibrium concentrations and those that measure a reaction&#8217;s kinetics. In case, the reaction must be at equilibrium at the time of measurement.
The method of determining the equilibrium concentrations depends on the desired sensitivity and ease of signal detection. For these reasons, spectroscopic assays are most widely used. In these experiments, the reaction produces an absorbance change of a reactant or a product at a given wavelength, detectable by a UV-Vis spectrophotometer. Alternatively, the reactant or product can be tagged with a fluorescent probe or may contain an intrinsic fluorophore. Then, the reaction progress can be measured from the change in fluorescence. These assays are performed by varying one reactant&#8217;s concentrations while the rest of the experiment is held constant. The results can then be graphed and analyzed with various curve fitting methods.
Interactions between proteins and ligands are also studied using a variety of biochemical and spectroscopic techniques. Structural analysis, using X-ray crystallography and NMR spectroscopy, aids in predicting protein-ligand interactions through molecular simulations. Theoretical and computational approaches, such as protein-ligand docking studies, are used extensively to characterize the position and interactions of small molecule ligands, including drug candidates. Computer-aided drug design is a fast and low-cost alternative to accelerate the pace of conventional trial and error drug testing.

The Equilibrium Binding Constant and Binding Strength of Proteins

平衡结合常数和结合强度

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at ...

Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions

To begin the BFP experiment, turn on the microscope and light source. Place the chamber onto the main microscope stage. The next step is to install all three micropipettes of the BFP. The probe to the left is to grab an RBC. The target to the right is to grab the cell, and the helper to the lower right serves to grab a bead.
To install, use a micro-injector to backfill a micropipette with experimental buffer. Take the micropipette off the pipette holder, and hold it at a lower place to allow liquid to drip from the tip. Quickly, insert the micropipette into the holder tip, making sure no air microbubble gets into the micropipette during the insertion.
Tighten the holder screw, then, mount each pipette holder onto its corresponding micromanipulator. Push the micropipette towards the chamber so that their tips enter the chamber buffer area. Adjust the position of the micropipette, and find them under the microscope field of view.
Move around the chamber holder&#39;s stage to find the colonies of the three elements one-by-one. Adjust the position of the corresponding micropipette by turning the knobs of the manipulators to let the tip of the micropipette approach one cell or bead. Aspirate the cell or bead by adjusting the pressure inside the corresponding micropipette. All three micropipettes will capture their corresponding elements.
Move around the chamber holder stage to find an open space away from the colonies of the injected elements where the experiment will be performed.
Switch the microscope visual mode to visualize the image on the computer program. Move all three elements on the pipette tips into the program&#39;s vision field. Next, align the probe bead and the RBC and carefully maneuver the probe bead to the apex of the RBC. Now, briefly impinge the bead onto the RBC and gently retract.
Adjust the pressure of the helper micropipette to gently blow the bead away so that it would be left glued onto the RBC apex. Move the helper micropipette away, and align the target, and probe bead.
On the program, in the Vision Field window, use the program tools to measure the respective radii of the probe micropipette, the RBC, and the circular contact area between the RBC and the probe bead. These values allow for the estimation of the spring constant of the RBC by the equation listed in the text protocol.
After entering the desired RBC spring constant into the program, draw a horizontal line across the RBC apex which will yield a curve in the adjacent window indicating the brightness of each pixel along this line. Drag the threshold line to be at around half the depth of the curve.
Next, select the desired experiment mode. Set the parameters as desired. Click Start which allows the program to move the target pipette and drive the target in and out of contact with the probe.
Data collection will be performed in parallel which records the position of the probe bead in real-time. Stop the program by clicking on the button Stop Experiment at which time a window will pop out to allow saving the acquired data.

生物膜 Force 探针定量受体-配体相互作用

Protein-protein interactions

EoE Sub Articles

ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells

Use 50,000 transfected BW cells with their desired targets in a single well of a 96-well plate. Incubate the plate for 48 hours. Then, freeze the plate at -20 degrees Celsius.
Coat an ELISA plate with anti-Mouse IL-2 antibody. Place 0.05% micrograms of anti-mouse-IL-2 in a volume of 50 microliters of PBS in each well. Incubate the coated plate overnight at 4 degrees Celsius.
Use a brief incubation period at 37 degrees Celsius to thaw the frozen BW cells that were previously incubated with their targets. Then, centrifuge the plate, and transfer 100 microliters of the supernatant from each well to the pre-coated ELISA plate, and incubate at 37 degrees Celsius for 2 hours.
After this, add biotin anti-mouse-IL-2 to the ELISA plate. Then, after incubation and washing, add HRP-conjugated Streptavidin to the plate. After incubation and washing, add 100 microliters of the TMB substrate solution to each well of the ELISA plate. Finally, read the ELISA plate at 650 nanometers.

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

摘要

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此视频中的章节

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

滴定 ELISA 法测定受体配体相互作用的离解常数

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

蛋白质 - 配体相互作用采用差示扫描荧光法测定

基于 SELEX 的体外结合测定法，用于鉴定 RNA-蛋白质相互作用

配体结合位点

平衡结合常数和结合强度

生物膜 Force 探针定量受体-配体相互作用

基于 ELISA 的报告基因检测研究 BW 细胞中受体-配体相互作用

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

摘要

探索更多视频

此视频中的章节

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

滴定 ELISA 法测定受体配体相互作用的离解常数

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

基于 SELEX 的体外结合测定法，用于鉴定 RNA-蛋白质相互作用

配体结合位点

平衡结合常数和结合强度

生物膜 Force 探针定量受体-配体相互作用

基于 ELISA 的报告基因检测研究 BW 细胞中受体-配体相互作用

蛋白质 - 配体相互作用采用差示扫描荧光法测定