这部分工作是由院内研究项目由美国国立卫生研究院，美国国家癌症研究所癌症研究中心; NCI和黑腹滨鹬治疗之间的合作研究协议，美国癌症协会授予IRG 97-152-17 OT和联邦基金支持美国国家癌症研究所，美国国立卫生研究院，根据合同HHSN26120080001E。

1。细胞裂解液的制备该协议的目的是，粘附细胞，任何GFP融合蛋白表达。所需的细胞数可以从低至10 6到多达20×10 6细胞，这取决于蛋白质表达的水平上。例如，裂解的HEK细胞过表达GFP-STAT3的准备处理10 T75烧瓶中生长的细胞裂解液用1毫升至70％附近汇合。然而，这种裂解液稀释150倍，以提供最佳的荧光水平MST实验。根据调查的性质和细胞内的蛋白质的本地化强烈地依赖于细胞裂解协议。超声描述如下，如果使用清洁剂，是不可取的，因为蛋白质的不稳定性，可能是​​最好的选择。不同的添加剂可以被添加到的裂解缓冲液中，以防止蛋白质修饰反应：乙二胺四乙酸防止磷酸化，钒酸钠抑制蛋白酪氨酸磷酸酶，所以dium氟化物是一种丝氨酸/苏氨酸磷酸酶抑制剂。 <ol><li>准备裂解液。对于下面的组合物易于提取胞浆蛋白效果很好的25mM三羟甲基氨基甲烷盐酸，pH值8.0，和蛋白酶抑制剂混合物稀释100倍（Sigma-Aldrich公司P2714，由2毫米AEBSF，0.3μM抑肽酶，130μM乌苯美司， 1 mM的EDTA，14微米E-64，1微米亮肽素）。另外，对于减可溶性蛋白，可以使用商业的RIPA缓冲液，蛋白酶抑制剂鸡尾酒和非变性洗涤剂。将缓冲区保持在冰上。 </li><li>简要地用冰冷的PBS，用10毫升每T75烧瓶中的缓冲液洗涤细胞。细胞置于冰上5分钟，或直至细胞开始从烧瓶中分离。刮细胞与细胞刮刀，如果有必要分离。 </li><li>悬浮细胞在10毫升冰冷的PBS和转移到预冷的14毫升圆底离心管。 </li><li>沉淀细胞在4℃下以400〜600×g离心5分钟。结合细胞从几个瓶中，在这一点上我f需要。 </li><li>删除PBS上清，加入200μl冰冷的裂解缓冲液重悬沉淀，将悬浮液转移到一个预先冷的1.5毫升的Eppendorf管。 </li><li>细胞置于冰上，以减少局部过热。裂解细胞的3倍10秒的超声脉冲在30％幅度，用一个2-3毫米预冷尖。保持在表面之下的前端，以尽量减少起泡。使用洗涤剂时的缓冲区，并在冰上孵育30分钟，而不是省略这一步。 </li><li>正确的裂解液，如果需要使用5 M氯化钠含有生理浓度盐（氯化钠100毫米）。 </li><li>通过离心分离收集溶胞产物于4℃在26000×g离心10分钟。 </li><li>确定最佳的溶胞产物稀释（在下面的第3节中所描述的）和所需的滴定（通常大约300μl的预稀释的溶胞产物）的细胞裂解液的量。 </li><li>等分试样的溶胞产物和存储在温度适合接受调查的蛋白质（-80℃，在大多数情况下）。 </li> &lt;/ OL&gt; 2。 MST缓冲器选择和准备<ol><li>由于蛋白 - 配体的相互作用依赖于缓冲液条件下，MST缓冲组合物的选择是基于一个特定系统的属性。它一般是有利的，至少两种不同的缓冲液进行测试。 </li><li>准备5倍MST缓冲区。我们有很好的经验，这两种成分：的HEPES（250毫米HEPES，pH值7.4; 25毫米氯化镁2，500 mM氯化钠，0.25％NP-40）和三羟甲基氨基甲烷盐酸（250毫米三羟甲基氨基甲烷盐酸，pH值7.4，氯化钠750毫米，50毫米氯化镁2 0.05％吐温20）。此外，BSA（结合混合物在最后的5％）可以帮助防止粘连蛋白塑料管和玻璃毛细管。 NaN 3的（0.5毫摩尔），也可以包括在内，以防止微生物的生长。 </li></ol> 3。优化裂解液稀释的测定<ol><li>选择LED的激励源，具有波长λ= 470nm的上MST仪器。 </li><li>加载毛细血管与CE会提取稀释2倍和10倍，MST缓冲。 </li><li>执行“查找毛细血管”的操作对MST仪器控制软件。在稀释的溶胞产物的最佳荧光范围是从400到1500荧光单位。 </li></ol> 4。的优化配体的浓度范围的测定<ol><li>的配位体的最高浓度应该是至少20倍高于预期的解离常数。 </li><li>最低的配位体的浓度应低于摩尔浓度的荧光蛋白。 </li></ol>请参阅配体浓度范围内估计的NanoTemper技术集中搜索工具。 5。细胞裂解液的制备和配体的稀释<ol><li>将需要数（通常为10-16）0.5毫升低吸附离心管冰管架。移取25μl的的MST缓冲液每根管子的底部。股票的配位体溶液中加入25μl的第一桶ë键（＃1，配位体的最高浓度），并进行串行使用的管的其余部分的配位体的2倍稀释。保持机架配体冰样品。 </li><li>慢慢地在冰上解冻细胞裂解液。 </li><li>与MST缓冲液稀释的细胞裂解物的荧光靶蛋白的结合反应中，以提供最佳水平。最终蛋白质浓度应该是接近预期的K D或更低。最终的解决方案中，应调整，以取得必要数量的荧光计数。 GFP-STAT3的溶胞产物中的浓度的测定，该仪器校准的荧光素与帮助。绿色荧光蛋白的溶胞产物中的摩尔浓度测定使用荧光素和绿色荧光蛋白的荧光量子产率，0.85和0.61的比例分别为15。 </li></ol> 6。微尺度热泳作用研究<ol><li>选择LED激发源，λ= 470纳米在MST仪器。 </li><li>地点0.5毫升低吸附管在TUBE机架对面配体连续稀释样品管。小心地加入15微升的细胞裂解物的每​​个管的底部。尽量不要接触管壁，避免样品损失。 </li><li>的配位体的样品中加入15μl的最高浓度（＃1）到相应的管＃1与细胞裂解物。拌匀，改变枪头。重复此步骤，除了最后一个，这应该不含有配位体与管的其余部分。 </li><li>最后一根试管中加入15μl的MST缓冲到拌匀。 </li><li>填补约2/3的第一毛细管从管＃1的结合混合物，向中心倾斜移动至该溶液，并将其放置在毛细管的毛细管托盘＃1的位置（最近的位置的纸盒开口） 。重复此步骤，与其余毛细血管。毛细管两端可以插入较长的实验用蜡。 </li><li>托盘放置里面MST仪器和关闭乐器的大门。 </li><li>执行“查找毛细血管”命令让仪器的毛细血管，并找到准确的位置，测量荧光的样品。 </li><li>的荧光信号强度的基础上，调整LED的功率（10-100％），使其到400-1,500个单位时间间隔。 </li><li>点击“开始”按钮，执行热泳实验。可以选择一个以上的IR-激光功率的试验，以便找到特定的系统的最佳温度梯度。收藏毛细血管为同一组中的从2-3的运行数据，以确保测量的可重复性。它要花费10-12分钟运行一组16个毛细管。 </li></ol> 7。微尺度的热泳数据分析<ol><li>打开分析软件。 </li><li>加载的项目文件夹。在出现的信息运行浏览器，选择收集在一个特定的红外激光功率热泳曲线。有一个选项，收集各种条件下，如红外激光功率打开所有热泳的痕迹，LED p奥尔，温度，浓度等 。切换和关闭一次，然后选择任何曲线分析，实验的名称（点击）。 </li><li>在的评价点数图“窗口中，选择T-跳热泳或热泳。确保正确定位，蓝色和红色的线条。要获得与标准偏差的平均点，选择“使用平均”或“区分运行”单独运行。 </li><li>要绘制的解离常数合适的，选择“一般”，进入并修复标记的分子浓度值（检查一个合适的窗口“菜单中的”集中“广场），贴合的曲线。其标准差的K D值出现在一个单独的弹出信息窗口。山法绘制一个合适的，选择“一般”，希尔法，然后贴合的曲线。 EC 50的亲和力在这种情况下，其标准偏差值。 杀敌或山“边界”的特征时，也可以利用饱和在绑定状态没有达到。 </li><li>保存在一个文本文件中，平均拟合数据传送到Excel。 </li><li>剧情F 范数 （归一化荧光），ΔF 范数 （如果不同的实验进行比较，在归一化荧光的差异），或分数绑定（见下面的公式）作为分子浓度的未标记的（滴定）的函数。 </li></ol>界的馏分（馏分在一个复杂的分子）=（千卡（C） - 未结合的）/（结合未结合的），其中，的千卡（C）是浓度C测定的热泳，未结合未结合的状态（当分子热泳在一个复杂的），并绑定是完全束缚态的热泳。

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作者宣称，他们有没有竞争经济利益。本出版物的内容并不一定反映卫生和人类服务部的观点或政策，也没有提及商号，商业产品，或组织由美国政府意味着赞同。

蛋白表达和纯化是一个费 ​​力和昂贵的步骤，然而，有必要由目前使用最多的方法测定相互作用的K D。 MST的应用可避免蛋白质纯化，从而大大简化和加快相互作用的定量表征。难以表达和纯化的蛋白质，如膜蛋白和转录因子的情况下特别显着的优点。 的主要限制和要求的MST是作为与绿色荧光蛋白融合的蛋白质的表达能力。然而，构建表达GFP融合人类和小鼠蛋白质?...

亲和力的分子间相互作用的定量表征是非常重要的生物医学研究在许多领域。约束力的解离常数（K D）是必不可少的，不仅在药物发现的，但也是一个重要的参数，表征任何生物系统中的任何二进制的相互作用。生化用于检测蛋白质-蛋白质相互作用的方法，如免疫沉淀和酵母双杂交筛选，不告知我们这些交互是多么紧张，而亲和定义，这是否存在特别复杂的给定条件下体内 。在药物发现过程中，结合法的发展是必要的，往往是最耗时的步骤之一。 杀敌测定最常用的方法包括荧光偏振，表面等离子体共振（SPR）技术，2放射配体结合，3温滴定量热平衡态，4透析（ED），超滤（UF），第5和超速离心（UC）。他们需要纯化的靶蛋白的数量显著。微量热泳（MST）是一个迅速发展的方法，该方法检测在一个微小的温度梯度定向的分子运动。任何更改的运动沿温度梯度的相对变化的生物分子导致的水合壳8，7 MST是用来确定结合亲和力，并已申请了荧光标记的蛋白质或荧光配体的配体结合研究靶蛋白。 9 MST允许直接在溶液中的相互作用的测量，而不需要固定到表面上（无固定技术）。实际上，任何有约束力的MST信号的变化是伴随着的大小变化，虽然从系统到系统的显着不同。对于检测MST分子运动，他们必须荧光NT。这种方法的主要限制，可以变成优势。如果在任何系统中作为GFP融合蛋白的表达，它是唯一的荧光分子，从而可以从细胞溶胞产物不经分离或无细胞表达系统研究。代约束条件允许以最少的文物的细胞裂解液中的主要挑战。在这里，我们描述了一个协议，可用于许多可溶性蛋白和膜蛋白的细胞裂解物的制备和MST实验。 STAT蛋白是潜在的胞质转录因子激活的酪氨酸磷酸化胞外信号响应和参与许多生物过程，包括免疫，造血，炎症，和发展10，在哺乳动物，STAT家族由STAT 1，2，3，4的，5A，5B，和6。所有激活的统计数据绑定到相同的DNA序列，所谓的气体图案，IFN-γ的激活序列。然而，transcri不同统计的ptional效果有很大的不同。11尽管在许多病理过程中的参与和广泛的研究，产生超过17,000出版物，K D STAT相互作用不同的DNA序列尚未确定。只有相对亲和力不同的统计变种天然气动机特点。11蛋白表达和纯化困难是表征统计的DNA结合选择性的主要障碍。虽然大多数研究都集中在“激活”的统计资料，这成了转录因子酪氨酸磷酸化，非磷酸化的统计（U-统计）转录调控作用的作用正在迅速崛起。 然而 ，这些机制的了解甚少，不清楚是否实际上U-统计与DNA结合，或通过与其他转录因子的相互作用行事。最近，我们已经表明，U-STAT3可以绑定到DNA顺序CES不同气图案具有更高的亲和力。13的发现具有重大意义，为我们理解这一重要蛋白质的生物学功能。我们应用微尺度热泳，确定相对亲和力STAT3气和AT丰富的寡核苷酸Ş+100（ 图4）。几乎相同的协议被用于不同的的STAT3的配位体，脂肽类抑制剂的结合在K D测定14否将关联的转录因子，作为阴性对照，使用GFP-STAT1结合从而证实选择性相互作用时，可检测到的。 14

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent/material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RIPA buffer</td>
 <td>Millipore</td>
 <td>20-188</td>
 <td>Other manufacturer's buffers work as well</td>
 </tr>
 <tr>
 <td>Protease inhibitors cocktail</td>
 <td>Sigma-Aldrich</td>
 <td>P2714</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>G008</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115 Capillary Tray</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>T001</td>
 <td></td>
 </tr>
 <tr>
 <td>Monolith NT.115 Standard Treated Capillaries</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>K002</td>
 <td></td>
 </tr>
 <tr>
 <td>NT Control software</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>2.0.2.29</td>
 <td></td>
 </tr>
 <tr>
 <td>NT Analysis software</td>
 <td>NanoTemper Technologies GmbH</td>
 <td>1.4.27</td>
 <td></td>
 </tr>
 <tr>
 <td>Table-top refrigerated centrifuge</td>
 <td>Eppendorf </td>
 <td>5417R</td>
 <td>Other microtube refrigerated centrifuges</td>
 </tr>
 <tr>
 <td>Protein LoBind Tube 0.5 ml</td>
 <td>Eppendorf </td>
 <td>22431064</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

protein purification-free method of binding affinity determination by microscale thermophoresis

蛋白质相互作用的定量表征是在几乎任何领域的生命科学，特别是药物发现至关重要。 K D测定所需要的现有的方法获得纯化的蛋白质，还代可以是费时和昂贵的。我们已经开发出一种测定结合亲和力的微尺度热泳（MST）协议，允许未经纯化的目标蛋白从细胞裂解液。该方法涉及过表达GFP的融合蛋白，在非变性条件下的细胞裂解。该方法应用到STAT3-GFP瞬时表达在HEK293细胞中的第一次，以确定具有不同序列的寡核苷酸的充分研究的转录因子的亲合性的。该协议是简单的，并且可以有各种不同的应用研究小分子，肽，DNA，RNA的蛋白质的相互作用，和对roteins。

测量非磷酸化STAT3的寡核苷酸结合蛋白的亲合性。 STAT3-GFP表达HEK293细胞，使用荧光标记的STAT3的DNA结合测定作为源。用RIPA缓冲液（20×10 6细胞/ ml）制备细胞裂解液。对于结合的研究，裂解物稀释150倍MST DNA的结合缓冲液中的荧光蛋白的结合反应（约20纳米），以提供最佳水平。未转染的HEK293细胞中已被用于评估的背景荧光，这被证明是不可探测的，即使在非...

Watch this Scientific Journal Video about Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis at JoVE.com

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

可广泛用于微型热泳（MST）无需纯化从细胞裂解液中的目标蛋白的结合亲和力的测定。该协议涉及过表达GFP融合蛋白，在非变性条件下的细胞裂解，MST信号检测在不同浓度的配体的存在下。

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

protein-purification-free-method-binding-affinity-determination

Research

JoVE Journal

Biology

30.3K 次观看.  National Cancer Institute. 可广泛用于微型热泳（MST）无需纯化从细胞裂解液中的目标蛋白的结合亲和力的测定。该协议涉及过表达GFP融合蛋白，在非变性条件下的细胞裂解，MST信号检测在不同浓度的配体的存在下。

视频: Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

科研

生物学

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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 ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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 ...

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 ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

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 ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...