Name: membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo
Uploaded: 2013-11-07T19:35:00
Description: Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

这项研究是由德意志研究联合会GraKo1121，Hu1011/2-1和SFB940到上海的资金支持

注： 表1是关于可在协议中表示缓冲区的详细信息。 1。蛋白质 - 蛋白质相互作用的甲醛交联的活细胞固定<ol><li>要开始准备的生长介质，原液和缓冲P1 <ol><li>制备500mL介质：该介质应该提供支持膜诱饵蛋白和猎物蛋白之间的相互作用的条件。此外，一个实验应该在没有相互作用，预期（ 例如，未经交联）的条件下进行。 注意：我们比较蛋白质 - 蛋白?...

<ol>
	<li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> J. Mol. Biol. 305, 567-580 (2001).</li><li>Bakheet, T. M., Doig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+identification+of+human+protein+drug+targets.">Properties and identification of human protein drug targets.</a> Bioinformatics. 25, 451-457 (2009).</li><li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat. Biotechnol. 25, (2007).</li><li>Daley, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly+of+membrane+proteins+into+complexes.">The assembly of membrane proteins into complexes.</a> Curr. Opin. Struct. Biol. 18, 420-424 (2008).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15, 118-124 (2012).</li><li>M&#252;ller, V. S., Jungblut, P. R., Meyer, T. F., Hunke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-SPINE:+An+improved+method+to+identify+protein-protein+interaction+partners+of+membrane+proteins+in+vivo.">Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo.</a> Proteomics. 11, 2124-2128 (2011).</li><li>Herzberg, C., 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=SPINE:+A+method+for+the+rapid+detection+and+analysis+of+protein-protein+interactions+in+vivo.">SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo.</a> Proteomics. 7, 4032-4035 (2007).</li><li>Sutherland, B. W., Toews, J., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+formaldehyde+cross-linking+and+mass+spectrometry+in+the+study+of+protein-protein+interactions.">Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions.</a> J. Mass Spectrom. 43, 699-715 (2008).</li><li>Hernandez, P., M&#252;ller, M., Appel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein+identification+by+tandem+mass+spectrometry:+Issues+and+strategies.">Automated protein identification by tandem mass spectrometry: Issues and strategies.</a> Mass Spectrom. Rev. 25, 235-254 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Hunke, S., Keller, R., M&#252;ller, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+the+Cpx-envelope+stress+system.">Signal integration by the Cpx-envelope stress system.</a> FEMS Microbiol. Lett. 326, 12-22 (2012).</li><li>Weber, R. F., Silverman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cpx+proteins+of+Escherichia+coli+K12+:+Structure+of+the+CpxA+polypeptide+as+an+inner+membrane+component.">The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component.</a> J. Mol. Biol. 203, 467-478 (1988).</li></ol>

膜脊柱的分析是生化的方法，使一个确认，并确定膜蛋白的这点未知的互动合作伙伴。膜脊柱由甲醛与链球菌标签膜诱饵蛋白的纯化结合在体内的交联。用免疫印迹法的组合有利于预测的相互作用伙伴的确认（ 图2）。此外，与MS分析的组合允许未知的相互作用伙伴的标识（ 图3）。对于这两种应用程序，有修改你的猎物蛋白质没有要求。此外，膜脊柱是足够的敏感...

了解蛋白质的功能，必须知道它的相互作用伙伴。几种传统的技术可用于可溶性蛋白的相互作用伙伴的标识。然而，这些技术是不容易转移到膜，由于其疏水性，4种蛋白。为了克服这个限制，我们已经开发了膜链球菌蛋白质相互作用实验（膜脊柱）6。它是根据脊柱的方法，它是唯一适合的可溶性蛋白7。 从两个优点的交联剂甲醛的膜-脊柱的好处：第一，甲醛可以轻易穿透细胞膜，因此产生一个活细胞8的相互作用组的精确快照。二，甲醛交联可煮沸9逆转。在这里，这两个优势是用来识别不仅永久性的，但也是短暂的膜保护制服的生产者价格指数插件6。 简言之，Strep-标签融合到完整的膜诱饵蛋白的C-末端。细胞中表达的膜蛋白诱饵孵育与甲醛的交联猎物蛋白质与膜诱饵蛋白（ 图1）。不需要猎物蛋白的修饰。接着，将膜部分准备。因此，膜蛋白是由洗涤剂处理和饵料蛋白质共同纯化，用亲和纯化它的猎物的蛋白质溶解。接着，将交联是通过煮沸颠倒，诱饵和其共洗脱猎物蛋白通过SDS-PAGE分离。最后，猎物的蛋白质可通过免疫印迹分析或质谱来识别。

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			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
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membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

膜蛋白是细胞存活必不可少的，因此是重要的治疗靶标1-3。由于他们在配合4个功能，方法来识别和描述它们之间的相互作用是必要的5。为此，我们开发了膜链球菌-蛋白相互作用的实验，称为膜脊柱6。该技术结合在体内使用可逆的交联剂甲醛与链球菌标签膜诱饵蛋白的亲和纯化的交联。在实验过程中，交联的猎物蛋白质是共纯化，用该膜诱饵蛋白，并随后通过煮沸分离。因此，两大任务可以分析蛋白质 - 蛋白质相互作用的膜蛋白（生产者价格指数），当使用膜脊柱执行：首先，提出一个合作伙伴的交互通过免疫印迹，并确认第二，新的互动合作伙伴通过质谱分析鉴定。而且，即使升嗷嗷的亲和力，瞬间PPIs是检测用这种技术。最后，膜脊柱是适用于几乎任何类型的细胞，使其适用于作为一个强大的筛选工具，以确定膜蛋白的生产者价格指数。

膜脊柱分析可以共同净化膜蛋白和瞬时相互作用蛋白的合作伙伴。共纯化是通过使用交联剂的甲醛来实现。两个参数是关键的，以防止非特异性交联：甲醛的浓度和交联时间。足够的，但不能过量使用甲醛可以很容易地通过免疫印迹法进行控制。甲醛交联的蛋白质复合物可以通过煮沸而不是由SDS处理进行分离。因此，它们可以在链球菌二型标签膜诱饵蛋白印迹如涂抹在免疫印迹的未煮沸样品的上?...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

一个生化方法描述识别体内蛋白质膜蛋白的相互作用（PPI）。该方法结合了蛋白质的交联，亲和纯化和质谱法，并且是适合于几乎任何类型的细胞或生物体。用这种方法，瞬态质子泵抑制剂的甚至识别成为可能。

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

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