<img src="/files/ftp_upload/1746/1746fig1.jpg" alt="图1" /> 图1的胱抑素B群（蓝色）（黄色）与木瓜蛋白酶的三维结构。突变的胱抑素乙残留以红色显示。 1。标签的自由互动使用Biacore系统的分析原则在一个典型的无标签结合使用Biacore系统的实验，生物分子被称为“配体”是连接到一个传感器芯片表面。系统带来的流动渠道接触到芯片表面，检测发生的，被称为“分析物”，其约束力的合作伙伴。当分析物的配体结合，积累在表面的质量变化是由表面等离子体共振（SPR）检测。 SPR响应约束力的分析物的量成正比。 由于绑定是在实时测量，动力学协会和为特定的相互作用的解离速率常数才能确定。从这些常量，它是可以计算的平衡解离常数的亲和力。它也可以计算出从稳态数据绑定的亲和力。类似的方法也可以用于确定特异性结合的配体表面的一种蛋白质的浓度。 2。一个蛋白质 - 蛋白质相互作用的动力学特性分析 Biacore X100，可以进行动力学分析，采用单周期动力学。在一个单周期的动力学实验，在一个单一的分析周期的分析物的浓度系列注入的，没有再生的表面之间的注射。因此，单周期动力学，使动力学分析时，很难找到合适的再生条件。 一旦已经收集了动力学实验数据，Biacore X100评估软件生成的数据交互模型拟合的K A，K D， 和 K ð值。 3。以确定蛋白质浓度的方法分析生物分子间的相互作用是很重要的，了解它们的功能。特征的蛋白质结合其他蛋白质，核酸，小分子生物化学研究的基础，并且发现在许多其他领域，包括药物发现的使用。 两个相互作用的蛋白质之间的相互作用动力学的精确测量，关键是要了解在实验样品分析物的使用的特异性结合蛋白的浓度。一个280分光光度计比色法或如读数用人布拉德福德试剂常用来确定总蛋白浓度。然而，蛋白质杂质影响的结果。更重要的是，蛋白质的活动和非活动的形式包括在总蛋白浓度。特别是在重组蛋白，可能无效，由于不正确折叠的情况下，重要的是，以确定样本中的特异性结合蛋白的百分比。 Biacore X100，涉及到具体的结合活性的浓度可以由具有约束力的响应水平相比，从已知的标准，或通过使用最近推出的方法无标定浓度分析中心（CFCA）的校准曲线。 CFCA不依赖于一个标准的。因此，中国金融认证中心是研究蛋白质的突变形式，标准通常是不可用的情况下特别有用。 在一个CFCA实验中，初始结合率是测​​量在不同流速条件下，当样品在芯片表面的扩散速率限制。分析物的扩散系数，是计算从最初的结合率 1,2特异性结合浓度时，考虑到流通池的尺寸和流速。 在动力学实验，分析物的浓度是用于计算动力学协会速率常数和实验数据的亲和力。 CFCA和Biacore系统的动力学测量都依赖于相同的互动性能。因此，使用浓度Biacore分析确定的特异性结合，而不是总蛋白浓度，增加了结果的可靠性。 4。利用Biacore分析表征之间胱抑素B和木瓜蛋白酶的相互作用这些蛋白质主要是在涉及nons哺乳动物胱抑素B是可逆的，有竞争力和紧束缚抑制蛋白的木瓜蛋白酶样半胱氨酸蛋白酶，主要是组织蛋白酶B，H，K，L和S。选修细胞内蛋白质的降解。被推定Cystatins不适当的水解细胞和组织，以保障这些酶。他们还从寄生虫和病毒灭活的半胱氨酸蛋白酶，可以参加对这类传染因子的入侵防御。此外，cystatins抑制几种植物半胱氨酸蛋白酶，如木瓜蛋白酶等，这是经常用来作为模型酶的结构与功能研究。胱抑素B群的立体结构与木瓜蛋白酶 3两种蛋白质之间的相互作用是由疏水性接触占主导地位，其中，从抑制剂方面，提供的N -和C -末端结束，两个发夹环（图1）。在这项研究中，我们研究的C -末端和胱抑素乙结合的第二次循环的重要性，为绑定使用牛胱抑素木瓜乙地区的互动与木瓜中含有点突变的变种。首先，我们确定的四个胱抑素B使用变种的特异性结合的浓度，以及野生型蛋白。积极胱抑素B的特异性结合浓度确定后，野生型和突变的变种结合，以木瓜蛋白酶半胱氨酸蛋白酶抑制剂乙的速度和亲和常数的测量使用Biacore X100。 5。仪器和试剂<ol><li> Cys3Ser/His75Gly胱抑素乙变种，Cys3Ser/Leu73Gly，Cys3Ser/Tyr97Ala和Cys3Ser产生如前面描述4。所有突变体包含一个额外的替代位置3丝氨酸，半胱氨酸，防止形成二硫键连接的非活动二聚体的胱抑素B。 </li><li>木瓜蛋白酶的目标还准备作为先前描述5。它的S -（甲硫基）木瓜蛋白酶（MMTS - 木瓜蛋白酶），有一个甲硫在活动间隙，使蛋白酶催化不活动组附加到Cys25。 </li><li> Biacore X100加包Biacore X100是用于测量和分析具体的有约束力的浓度和结合动力学。 </li><li> MMTS - 木瓜蛋白酶固定在传感器芯片CM5使用Biacore胺偶联试剂盒。 </li><li>运行在25 ° C，缓冲区是在pH 7.4 0.01 M羟乙基，0.15 M氯化钠（NaCl），0.0034 M EDTA，0.05％聚山梨酯20。 </li><li>彼此之间半胱氨酸蛋白酶抑制剂乙的变种，在传感器表面是再生在10μL/ min的流速为30秒与20毫米氢氧化钠。 </li></ol> 6.Immobilization的配体MMTS - 木瓜蛋白酶 CFCA分析MMTS - 木瓜蛋白酶共价结合中国金融认证中心的依赖率分析物分子的表面扩散（大众运输的限制）的限制条件下的结合率的测量。这是由配体的高固定水平的青睐。 MMTS - 木瓜蛋白酶的固定化是建立和运行使用固定Biacore X100控制软件向导。 <ol><li>激活的琥珀酰亚胺（NHS）和碳二亚胺（EDC）为10μL/ min的流速在7分钟的混合物注入流通池2的表面。流单元1保持不变，以作为参考表面。 </li><li>在50μg/ml注入MMTS - 木瓜蛋白酶在pH 4.5的醋酸钠缓冲液5μL/ min的流速在15分钟。 </li><li>乙醇胺是在流速10μL/ min的取消余下的积极酯注射7分钟。 </li></ol>整个耦合过程的结果〜3000 RU MMTS - 木瓜蛋白酶固定在流通池2。 共价结合的MMTS - 木瓜蛋白酶的动力学分析用于固定MMTS - 木瓜蛋白酶的动力学分析，唯一的区别是，动力学分析，固定层次的需求要低，以避免结合率，成为传播有限公司，同样的程序。流单元1留在这一步中没有修改，以及以作为参考表面。 <ol start="4"><li> MMTS - 木瓜蛋白酶激活与NHS和EDC在7.1的混合物表面后，被注入在1μg/ml为50秒。后，表面上是在步骤7.3乙醇胺注射激活。 </li></ol>耦合度应约50 RU此过程后。 7。确定胱抑素B的浓度，使用中国金融认证中心检测<ol><li> CFCA实验是根据CFCA Biacore X100加包向导成立。约10纳米的蛋白注入流速10和100μL/分钟，一式两份，每份为48秒。每个周期之间，表面上是20毫米氢氧化钠再生。 </li><li>包括为每个流率的空白注射。 </li><li>蛋白浓度，然后确定从数据绑定，使用次（图3）中国金融认证中心发送Biacore X100加包评估软件的评价功能。 <img src="/files/ftp_upload/1746/1746fig2.jpg" alt="图2" /> 图2。CFCA野生型半胱氨酸蛋白酶抑制剂B.特异性结合的浓度数据分析的结果是在10和100μL/ min的流速获得sensorgrams提取。 <img src="/files/ftp_upload/1746/1746fig3.jpg" alt="图3" /> 图3突变体。浓度测定， 使用 280测量（N = 3）和中国金融认证中心（N = 2） 。误差线表示标准误差。摩尔吸光系数计算（6）中所述。可以观察到一个由两种方法确定的Cys3Ser/Leu73Gly突变的浓度值相差较大。 </li><li>当比较一个由中国金融认证中心，280测量确定的浓度，它是明确的，而在大多数情况下的蛋白质是完全积极的Cys3Ser/Leu73Gly变种，活性蛋白的比例是非常低（图3 ）。下表显示了胱抑素B相关的总浓度由280变体的活动。 <table border="0"><tbody><tr><td>样品</td><td>中国金融认证中心在280（％） </td></tr><tr><td>野生型</td><td> 94 </td></tr><tr><td> Cys3Ser/His75Gly </td><td> 101 </td></tr><tr><td> Cys3Ser/Leu73Gly </td><td> 9 </td></tr><tr><td> Cys3Ser/Tyr97Ala </td><td> 99 </td></tr><tr><td> Cys3Ser </td><td> 83 </td></tr></tbody></table></li></ol> 8。测量结合，以木瓜蛋白酶胱抑素乙动力学<ol><li>中国金融认证中心测量的基础上，编写了两方面的的一系列浓度从2.5至40纳米，每个胱抑素乙变种。 </li><li>动力学实验是在Biacore X100使用与单周期方法的动力学向导成立。表面不再生之间的注射，但每个分析周期结束后使用再生液20毫米氢氧化钠。 </li><li>成立，使每个周期包含示例是一个空循环，缓冲区，而不是样品注入两侧的实验。 </li><li>使用Biacore X100动力学评价功能，自动履行职权的空白减法减去装修前以1:1的交互模型的数据。 </li><li>胱抑素乙变种结合木瓜蛋白酶获得的实验数据如图4所示。下表总结了协会和分解速率常数和动力学分析得到的平衡解离常数。 <table border="0"><tbody><tr><td>样品</td><td> K A（M -1 s - 1时） </td><td> k ð（-1） </td><td> ð K（男） </td></tr><tr><td>野生型</td><td> 1.8 × 10 6 </td><td> 0.41 x 10 -3 </td><td> 2.3 × 10 -10 </td></tr><tr><td> Cys3Ser/His75Gly </td><td> 1.1 × 10 6 </td><td> 1.7 × 10 -3 </td><td> 1.5 × 10 -9 </td></tr><tr><td> Cys3Ser/Leu73Gly </td><td> 1.1 × 10 6 </td><td> 23 × 10 -3 </td><td> 2.2 × 10 -8 </td></tr><tr><td> Cys3Ser/Tyr97Ala </td><td> 1.7 × 10 6 </td><td> 12 × 10 -3 </td><td> 7.1 × 10 -9 </td></tr><tr><td> Cys3Ser </td><td> 0.9 × 10 6 </td><td> 0.53 x 10 -3 </td><td> 5.8 × 10 -10 </td></tr></tbody></table> <img src="/files/ftp_upload/1746/1746fig4.jpg" alt="图4" /> 图4。胱抑素乙变种MMTS -木瓜蛋白酶的结合动力学配 ​​置文件确定使用单周期动力学。 Sensorgrams显示空白和参考减去动能适合1:1的互动模式覆盖在黑色的数据。 </li><li>协会率的突变体与野生型蛋白（图5，上游面板），解离速率常数的突变体，可以接近两个数量级，与相应的平衡解离增加固定（图5，下半部分）。 <img src="/files/ftp_upload/1746/1746fig5.jpg" alt="图5" /> 图5。率和亲和常数的计算是基于从中国金融认证中心获得的浓度。 K A，K ð K ð与相关的基因突变，变化是描绘在图形。 </li><li>它是由于样品浓度是在从实验数据的关联率不断的计算参数，重要的是，它是正确的的。使用CFCA测量，而不是一个280的浓度会得出这样的结论，Leu73协会率，其中更换似乎导致约10倍速度比其他半胱氨酸蛋白酶抑制剂乙变种协会是非常重要的。然而，特异性结合浓度测量由中国金融认证中心是在这种情况下，总蛋白，只有约10％。这是考虑到这一点变得清晰Leu73协会率是类似的其他变种。因此，中国金融认证中心允许K A和 K ð从而使可能的互动机制的适当的解释正确评估。 </li></ol>

<ol>
	<li>Christensen, L. L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+protein+concentration+determination+using+biosensor+technology+under+conditions+of+partial+mass+transport+limitation.">Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.</a> Anal. Biochem. 249, 153-164 (1997).</li><li>Sigmundsson, K., M&#226;sson, G., Rice, R., Beauchemin, N., &#214;brink, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+active+concentrations+and+association+and+dissociation+rate+constants+of+interacting+biomolecules:+an+analytical+solution+to+the+theory+for+kinetic+and+mass+transport+limitations+in+biosensor+technology+and+its+experimental+verification.">Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification.</a> Biochemistry. 41 (26), 8263-8276 (2002).</li><li>Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., Turk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+refined+2.4+A+X-ray+crystal+structure+of+recombinant+human+stefin+B+in+complex+with+the+cysteine+proteinase+papain:+a+novel+type+of+proteinase+inhibitor+interaction.">The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.</a> EMBO J. 9 (6), 1939-1947 (1990).</li><li>Pol, E., Bj&#246;rk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+second+binding+loop+and+the+C-terminal+end+of+cystatin+B+(Stefin+B)+for+inhibition+of+cysteine+proteinases.">Importance of the second binding loop and the C-terminal end of cystatin B (Stefin B) for inhibition of cysteine proteinases.</a> Biochemistry. 38 (32), 10519-10526 (1999).</li><li>Bj&#246;rk, I., Pol, E., Raub-Segall, E., Abrahamson, M., Rowan, A. D., Mort, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+changes+in+the+association+and+dissociation+rate+constants+for+binding+of+cystatins+to+target+proteinases+occurring+on+N-terminal+truncation+of+the+inhibitors+indicate+that+the+interaction+mechanism+varies+with+different+enzymes.">Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes.</a> Biochem. J. 299, 219-225 (1994).</li><li>Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., Gray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+the+molar+absorption+coefficient+of+a+protein.">How to measure and predict the molar absorption coefficient of a protein.</a> ProteinScience. 4 (11), 2411-2423 (1995).</li></ol>

作者是受聘于GE医疗产生本文中使用的试剂和仪器。

在这项工作中，四突变体和野生型半胱氨酸蛋白酶抑制剂乙生产的，以评估的第二约束力循环的重要性和C -端之间胱抑素B和木瓜蛋白酶的相互作用结束。这项研究表明优势和易于使用Biacore X100，以确定具体的的具有约束力的浓度和蛋白质相互作用的动力学分析，以了解结构与功能的关系。我们发现，测量总蛋白浓度不露的蛋白质结合活性降低，在这种情况下引入了一个明显的测量误差在确定结合的亲和力和动力学的变种。胱抑素乙变种的特异性结合的浓度进行了测定与Biacore X100使用中国金融认证中心。由中国金融认证中心作为输入的动力学分析使用浓度测量结果可靠率和亲和常数，从而使正确的解释的互动机制。 的突变体的亲和力下降几乎完全是由于增加 k D值， 而 k的影响只是轻微。这种行为表明，第二约束力环区和C末端的木瓜蛋白酶抑制剂的结合率的重要。相反，它们有助于亲和力，主要通过保持连接酶抑制剂，已经形成了复杂的一次。

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the importance of correct protein concentration for kinetics and affinity determination in structure-function analysis 

在这项研究中，我们探索牛半胱氨酸蛋白酶抑制剂胱抑素B和木瓜蛋白酶（图1），植物半胱氨酸蛋白酶的催化活性的形式之间的相互作用，通过实时的无标签使用Biacore X100的分析。几个点突变与木瓜蛋白酶的相互作用等方面的半胱氨酸蛋白酶抑制剂与乙的变种，是生产。对于每个胱抑素乙变种，我们确定其特异性结合的浓度，使用无标定的浓度分析中心（CFCA）和总蛋白浓度获得一个确定的值比较<sub&gt; 280</sub&gt;。在此之后，每个结合，以木瓜蛋白酶半胱氨酸蛋白酶抑制剂乙变种的动力学测量采用单周期动力学（SCK）。我们表明，我们研究的四个半胱氨酸蛋白酶抑制剂乙变种一个有约束力的局部活跃。由中国金融认证中心透露，这部分的活动，转化为在该协会的速率常数显着性差异（<em&gt; K</em<sub“一</sub&gt;）和亲和力（<em&gt; K</em<sub&gt; D</sub&gt;）相比，使用总蛋白浓度计算值。使用中国金融认证中心结合在一个结构与功能的研究动力学分析，有助于获得可靠的结果，并有助于互动机制，使正确的解释。

Watch this Scientific Journal Video about The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis at JoVE.com

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

我们采用无标记蛋白质相互作用分析利用Biacore X100几个胱抑素乙突变体的结合，木瓜蛋白酶，通过动力学特性的结构与功能分析。无标定浓度分析中心（CFCA）的措施，而不需要为标准曲线与保留结合活性的蛋白浓度。我们表明，浓度使用CFCA增加的动力学分析的可靠性，并能够可靠地确定，即​​使重组蛋白的活性降低，动力学常数确认。

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), ...

the-importance-correct-protein-concentration-for-kinetics-affinity

Research

JoVE Journal

Biology

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

20.4K 次观看.  GE Healthcare Bio-Sciences AB. 我们采用无标记蛋白质相互作用分析利用Biacore X100几个胱抑素乙突变体的结合，木瓜蛋白酶，通过动力学特性的结构与功能分析。无标定浓度分析中心（CFCA）的措施，而不需要为标准曲线与保留结合活性的蛋白浓度。我们表明，浓度使用CFCA增加的动力学分析的可靠性，并能够可靠地确定，即​​使重组蛋白的活性降低，动力学常数确认。

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

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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

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

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

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

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

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

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

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 ...

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 ...

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

现货正确的组织多组织块每一次

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...