Name: an in vivo crosslinking approach to isolate protein complexes from drosophila embryos
Uploaded: 2014-04-23T21:00:00
Description: Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

我们感谢乔丹·戴维斯，岩岩林，埃里克·特德和Jimiao郑为他们的技术帮助，这项研究。这项工作是支持由美国国家科学基金会CAREER授予MCB-1054962，以ALA

1，准备大苹果汁琼脂平板<ol><li>为了使4片，加375毫升H 2 O，11.25克飞琼脂和搅拌棒的1000ml的烧瓶中。这是驴友A.高压灭菌混合物A与烧瓶盖子松松地盖上瓶盖在一个30分钟灭菌循环液体货物。 </li><li>添加125毫升苹果汁，12.5克蔗糖和搅拌棒，以500毫升的烧杯中。这是混合B.热混合物B在加热平台，同时搅拌并保持温度在约70℃，直至混合物A的高压灭菌完成。 </li><li>在完成混合的高压灭菌，转?...

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L., Ramos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+RNA-protein+granules:+insight+from+the+germline.">Building RNA-protein granules: insight from the germline.</a> Trends in cell biology. 20, 482-490 (2010).</li><li>Gao, M., Arkov, A. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. , (2001).</li><li>Thomson, T., Liu, N., Arkov, A., Lehmann, R., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+new+polar+granule+components+in+Drosophila+reveals+P+body+and+ER+associated+proteins.">Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins.</a> Mechanisms of development. 125, 865-873 (2008).</li><li>Vasilescu, J., Guo, X., 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=Identification+of+protein-protein+interactions+using+in+vivo+cross-linking+and+mass+spectrometry.">Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.</a> Proteomics. 4, 3845-3854 (2004).</li><li>Klockenbusch, C., 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=Optimization+of+formaldehyde+cross-linking+for+protein+interaction+analysis+of+non-tagged+integrin+beta1.">Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1.</a> J Biomed Biotechnol. 2010, 9275-9285 (2010).</li><li>Nowak, D. 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甲醛已被普遍用作交联试剂用于鉴定蛋白质 - 蛋白质和蛋白质 - 核酸相互作用。良好的溶解性和细胞膜的通透性，与下游质谱程序的兼容性在一起，使甲醛的理想人选剂胞内交联应用3,15〜17。特别是，它被成功地用于鉴定与瓦萨，在果蝇 11的临界生殖细胞RNA解旋酶相关的mRNA。此外，甲醛具有最短间隔臂之一（2.3 - 2.7 a）在市售的交联剂，这意味着只存在于邻近的分子会在?...

的多亚基蛋白装配和DNA或RNA-蛋白质复合物的分离进行识别的蛋白质复合物，通过DNA-结合调节蛋白或RNA结合蛋白的RNA靶标确认的基因位点。不同的方法允许通过具有给定的RNA结合蛋白（CLIP-SEQ）2相关的转录因子或染色质蛋白（芯片起）1和RNA靶标确认的DNA位点的全基因组鉴定。的RNA衍生的cDNA或DNA靶库，然后深深的测序。这些方法使用化学或紫外线诱导的交联，以与针对所研究的复合物的蛋白组分的抗体稳定后跟免疫沉淀（IP）的配合物。 在一个生物体的发展，许多蛋白质​​复合物形成短暂。因此，关键的是要分析这些复合物在体内的组成和功能，了解控制dev的分子机制elopment。这样的体内分析将优于在体外的方法，因为这几乎是不可能重现交互的组件和细胞生物化学环境体外的天然浓度。在这里，我们表明，我们成功地使用从果蝇胚胎中分离大蛋白复合物在体内的方法。在该方法中，蛋白质复合物中的活胚交联与甲醛的低浓度，其后的兴趣蛋白质复合物被分离出的IP与抗体对复合物随后的复合物和质谱分析的凝胶纯化的已知成分识别未知的复杂组件。由于甲醛是能够渗透细胞膜并具有2.3-2.7埃3的交联范围内，蛋白质复合物可被交联的体内和复杂的组件都可能是彼此接近的。在这篇文章中，我们采用都铎（TUD）蛋白复合物的分离作为一个例子说明这种方法。 TUD是一个种系的蛋白这是种系发育4-7必不可少的。该蛋白含有11已知与其他多肽8-10的甲基化精氨酸或赖氨酸互动TUD域。 以前，我们已经产生的转基因果蝇行其表达HA-标记的功能TUD 5，因此，特定的抗HA抗体用于拉下TUD复杂交联后。 除了蛋白质 - 蛋白质交联，甲醛可产生核酸 - 蛋白质交联，并用于在芯片起实验。此外，在果蝇中 ， 在体内与甲醛交联已经允许瓦萨RNA解旋酶蛋白11的RNA靶的鉴定。 虽然本文中，我们描述了一种用于体内交联和PUR蛋白质复合物从果蝇胚胎的ification，这种方法可以适用于其它生物体和组织。

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

许多细胞过程是由多亚基蛋白复合体的控制。经常这些复合物形成短暂且需要本地环境来组装。因此，能够识别这些功能性蛋白质复合物，其以它们稳定在细胞裂解和随后的纯化前体是很重要的。在这里，我们描述了用于分离大的善意蛋白复合物从果蝇胚胎的方法。此方法是基于使用的甲醛浓度低，它可以很容易地穿过细胞膜由体内交联的胚胎内的复合物的胚胎透化和稳定化。随后，感兴趣的蛋白质复合物被免疫纯化后凝胶纯化并通过质谱法进行分析。我们使用的铎蛋白复合物，它是种系发育必不可少纯化说明这种方法。都铎王朝是一个大的蛋白质，它包含多个域的都铎王朝 - 小模块与靶蛋白的甲基化精氨酸或赖氨酸互动。这个方法可以适用于天然蛋白质复合物从不同的生物体和组织的隔离。

7％凝胶步骤（ 图1中示出），然后通过蛋白质印迹（ 图2） -交联的效率和交联TUD蛋白复合物的成功的纯化的SDS-PAGE上的3％进行了分析。 使用3％的目的 - 7％步骤凝胶是基于交联TUD蛋白复合物从复合物中剩余的未交联的TUD蛋白和浓度的有效分离。根据上面提到的我们的体内交联条件，TUD蛋白的相当大的比例可能仍然未交联的。这个自由TUD蛋白?...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

多组分蛋白复合物的细胞功能和发展过程中发挥关键作用。在这里，我们描述了用于从果蝇胚胎分离的天然蛋白质复合物后，随后交联的复合物用于随后的结构-功能分析的纯化在体内交联的方法。

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Research

JoVE Journal

Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

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 ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Upright Imaging of Drosophila Embryos

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...