Name: real time measurements of membrane protein:receptor interactions using surface plasmon resonance (spr)
Uploaded: 2014-11-29T14:00:00
Description: Watch this Scientific Journal Video about Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR at JoVE.com

Research at the Lewinson lab is supported by grants from the Israel Academy of Science, the Rappaport Institute for Biomedical research, the Meriuex Research Foundation, and the Marie Curie career reintegration grant (OL). EV is supported by the Fine scholarship and by the Technion Faculty of Medicine. NLL is supported by grants from the Israel Academy of Science.

1.蛋白质样品和缓冲液准备<ol><li>样品制备：纯化目的蛋白，并确保所有聚集体被除去，通过注入实验前的蛋白上的凝胶过滤柱或通过超速离心（通常为10分钟，在100000×g离心就足够了）。 注：虽然可取，高纯度蛋白制剂不是必须的。 SPR已成功地用于与低于完美纯化，甚至与总提取物8,9。 </li><li>缓冲液配制： <ol><li>制备实验的缓冲液（称为“运行缓冲液”，或包）。对于这里描述?...

<ol>
	<li>Rich, R. L., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+surface+plasmon+resonance+biosensor+analysis.">Advances in surface plasmon resonance biosensor analysis.</a> Current Opinion in Biotechnology. 11, 54-61 (2000).</li><li>Rich, R. L., Myszka, D. G. H. i. g. h. e. r. -. t. h. r. o. u. g. h. p. u. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=label-free,+real-time+molecular+interaction+analysis.">label-free, real-time molecular interaction analysis.</a> Analytical Biochemistry. 361, 1-6 (2007).</li><li>Helmerhorst, E., Chandler, D. J., Nussio, M., Mamotte, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+and+label-free+bio-sensing+of+molecular+interactions+by+surface+plasmon+resonance:+a+laboratory+medicine+perspective.">Real-time and label-free bio-sensing of molecular interactions by surface plasmon resonance: a laboratory medicine perspective.</a> The Clinical Biochemist Reviews. 33, 161 (2012).</li><li>Kyo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MafG+interaction+with+Maf+recognition+element+arrays+by+surface+plasmon+resonance+imaging+technique.">Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique.</a> Genes to Cells. 9, 153-164 (2004).</li><li>Katsamba, P. S., Park, S., Laird-Offringa, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+studies+of+RNA&#8211;protein+interactions+using+surface+plasmon+resonance.">Kinetic studies of RNA&#8211;protein interactions using surface plasmon resonance.</a> Methods. 26, 95-104 (2002).</li><li>Vigonsky, E., Ovcharenko, E., Lewinson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+molybdate/tungstate+ABC+transporters+that+interact+very+differently+with+their+substrate+binding+proteins.">Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins.</a> Proc. Natl. Acad. Sci. U S A. 110, 5440-5445 (2013).</li><li>Lewinson, O., Lee, A. T., Locher, K. P., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation.">A distinct mechanism for the ABC transporter BtuCD-BtuF revealed by the dynamics of complex formation.</a> Nat. Struct. Mol. Biol. 17, 332-338 (2010).</li><li>Kyo, M., Usui-Aoki, K., Koga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Detection+of+Proteins+in+Crude+Cell+Lysate+with+Antibody+Arrays+by+a+Surface+Plasmon+Resonance+Imaging+Technique.">Label-Free Detection of Proteins in Crude Cell Lysate with Antibody Arrays by a Surface Plasmon Resonance Imaging Technique.</a> Analytical Chemistry. 77, 7115-7121 (2005).</li><li>Mori, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+protein+kinase+activities+of+cell+lysates+using+peptide+microarrays+based+on+surface+plasmon+resonance+imaging.">Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging.</a> Analytical Biochemistry. 375, 223-231 (2008).</li><li>Mol, N., Fischer, M. E., Mol, N. J., Fischer, M. J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Plasmon Resonance Vol. 627 Methods in Molecular Biology. 1, 1-14 (2010).</li><li>Van Der Merwe, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface plasmon resonance. Protein&#8211; ligand interactions: hydrodynamics and calorimetry. , 137-170 (2001).</li><li>Lewinson, O., Lee, A. T., Locher, K. P., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+mechanism+for+the+ABC+transporter+BtuCD-BtuF+revealed+by+the+dynamics+of+complex+formation.">A distinct mechanism for the ABC transporter BtuCD-BtuF revealed by the dynamics of complex formation.</a> Nat. Struct. Mol. Biol. 17, 332-338 (2010).</li></ol>

SPR是一种研究分子相互作用高度敏感的方法，并且经常是，它提供了短暂的（但重要）的相互作用的实时监测的唯一方法。一个例子是瞬时相互作用本文中所呈现，不能由任何其他方法（下拉测定法，脂质体沉淀测定法6）来检测。此外，虽然其他方法被限制为平衡测量（不论数量或质量），SPR是衡量也预平衡动力学的唯一技术之一。 其高度的敏感性也是它的警告。?...

蛋白质 - 蛋白质相互作用（PPI）;的形成和蛋白复合物的解离，在许多生物过程（ 如复制，转录，翻译，信号传导，细胞-细胞通讯）的重要事件。通常执行使用下拉或免疫沉淀实验PPI的半定量研究。但是，这些（以及类似）的技术在相关度的可以测量的范围内（低微摩尔和更高的亲和力）由于在洗涤步骤所固有的这些技术的限制。这种“终点”技术不能识别往往是巨大的生物后果短暂的或低亲和力的互动。此外，这种方法的时间分辨率是非常有限的，通常要比反应的速率较慢的订单。 SPR克服了这些缺点，由于其高度的灵敏度和优异的时间分辨率1,2。 SPR是一种无标记方法，因此分子间相互作用可以测量（只要该质量变化，可以检测出）。除了​​PPI，它已被广泛地用于表征蛋白质-配体，蛋白质-药物（或小分子），蛋白质-DNA和蛋白质-RNA相互作用3-5。结果记录并绘制在实际的时间，这使得实验条件和灵活的实验设计的快速修改。 后面的SPR基于票据的物理原理是测量在传感器表面上的折射率的改变时的质量变化2的光学系统的利用率。之一的相互作用伙伴（以下称配体）被固定到聚合物基质芯片和第二分子（以下称分析物）被流过的表面上。分析物结合至该配体改变在芯片表面的质量。这种质量变化是直接按比例变化有关的表面的折射率。该结果绘于实时和呈现为响应单位（RU），为时间的函数。这样的情节被称为sensogram（ 例如 ， 图1）。由于SPR如下的相互作用的完整的时间过程中，预平衡动力学速率常数推导，除了平衡亲和力。如下面详细描述的，一个给定的系统的预均衡行为持有多的信息，并且提供了一个非常不同的角度比单独的平衡。一旦系统被校准，实验是非常快的，并且只需要少量的蛋白材料（微克至纳克量）。收集一个给定的系统的完整动力学信息可以在几天来实现。 SPR的高灵敏度的测量能提供使用的任何其它技术6那是不可能的。然而，这种高灵敏度是一个&#39;双刃剑&#39;，因为它可以为误报数据的主要来源。影响的反射率的任何因素被记录并绘制在sensogram。因此，APpropriate控制必须用来消除误报，并补充办法支持高度建议。 图1示出使用NTA被覆传感器芯片的SPR实验的进展。在面板A中的sensogram示出了喷射的镍离子在NTA基质（未消减数据），并减去从所述阴性对照细胞产生的信号后面板的BD显示数据。红色和蓝色的箭头表示注射的开始和结束，分别。配位体上的芯片固定逐渐改变质量，直到喷射终止。终止配体的注射后观察到的稳定的高原反映配体与所述表面（ 图1B，周期2）的稳定的缔合。一旦一个稳定的基线实现的缓冲器注射在配位体和所述参考单元（ 图1B，周期3）。这注射作为&#39;空白对照“，并且将在分析过程中减去。在注射后，小的变化被记录，反映了质量通过芯片的流动。然后在一个单独的循环（ 图1B，周期4）中，分析物被注入;在RU的逐步增加代表分析物的关联配体。所述结合位点变得逐渐占据直至达到平衡。只要注射结束后，在个RU的下降反映了复杂的解离。从这样的传感图的预均衡和平衡速率常数可以衍生（见后面）。 图1示出了配体和分析物之间的瞬时相互作用。当该相互作用是更稳定的，在RU水平的下降是比较慢，这反映了较低的kð。 这里，我们描述一个SPR实验旨在表征膜转运（洗涤剂溶解）和它的功能性配偶体之间的相互作用，其同源底物结合蛋白6,7。所选择的MOD埃尔系统是一个ATP结合盒（ABC）转运，Archeaglobus fulgidus的ModBC-A。这个系统被选定为它的高度可重复性的结果，高信噪比，和古典&#39;开/关&#39;速率。此外，同源物ABC转运可作为重要的阴性对照。转运，ModBC（配位体A）中，从使用的洗涤剂，纯化和固定到芯片上的膜萃取。其可溶性互动的合作伙伴，MODA，是分析物。作为阴性对照配体，不同的ABC转运RbsBC被使用（“配体B”）。

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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Biacore T200</td>
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			<td style="width:119px;">28-9750-01</td>
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			<td height="21" style="height:21px;width:209px;">Sensor Chip NTA&#160;</td>
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			<td style="width:119px;">14100532</td>
			<td></td>
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			<td height="21" style="height:21px;width:209px;">BIAevaluation</td>
			<td style="width:107px;">GE Healthcare</td>
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			<td height="21" style="height:21px;width:209px;">Biacore T200 Software&#160;</td>
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			<td height="21" style="height:21px;width:209px;">Nickel chloride</td>
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			<td height="21" style="height:21px;width:209px;">Sodium tungstate dihydrate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">T2629</td>
			<td></td>
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			<td height="21" style="height:21px;width:209px;">Sodium Chloride&#160;</td>
			<td style="width:107px;">Bio-Lab LTD.</td>
			<td style="width:119px;">19030591</td>
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			<td height="21" style="height:21px;width:209px;">Trizma base</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">T1503</td>
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			<td height="63" style="height:63px;width:209px;">Ethyldiamine-tetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">E5134</td>
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			<td height="41" style="height:41px;width:209px;">n-Dodecyl-&#946;-D-Maltopyranoside (DDM)</td>
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			<td height="42" style="height:42px;width:209px;">Sodium dodecyl sulfate solution&#160;</td>
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			<td style="width:119px;">05030</td>
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			<td height="21" style="height:21px;width:209px;">Kimwipes KIMTECH</td>
			<td style="width:107px;">Kimberly-Clark</td>
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			<td height="21" style="height:21px;">Nylon (NY) Membrane Filter</td>
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			<td height="21" style="height:21px;width:209px;">ModBC ABC transporter</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:209px;">RbsBC ABC transporter</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:209px;">ModA Substrate Binding Protein</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
			<td></td>
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real time measurements of membrane protein:receptor interactions using surface plasmon resonance (spr)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

在本文所描述的系统中，一个NiNTA芯片被用来固定所述His-标记的膜转运6,7。作为同型二聚体，各转运被双重标记，提高其结合到NiNTA芯片。继镍负载，配体A（感兴趣的转运）被固定在FC = 2，多达〜3500 RU（协议周期2， 图2A黑色标签）。然后，用同样的流量和喷射持续时间，配体B（对照配体）被注射到的Fc = 1，最初仅达到多达〜3000 RU。确保相似的蛋白质的量被固定在这两个流?...

Watch this Scientific Journal Video about Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR at JoVE.com

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

表面等离子共振（SPR）是一种无标记检测方法，实时生物分子相互作用。在这里，协议的膜蛋白：受体相互作用实验说明，在讨论该技术的优点和缺点。

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

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