Name: medium-scale preparation of drosophila embryo extracts for proteomic experiments
Uploaded: 2017-05-30T15:00:00
Description: Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

作者感谢Veraksa实验室的成员对手稿的帮助意见和协议改进建议。 AV由NIH授权GM105813和NS096402支持。 LY得到马萨诸塞州大学波士顿赛诺菲药学博士学位的支持。

5.制备5L飞行笼（适合15cm板） <ol><li>获得所需材料：带盖的5夸脱（4.7升）容器（ 图1A ），尼龙网和剃刀刀片。 </li><li>标记一个12厘米直径的圆圈，并使用剃刀刀片在容器底部切开一个孔（ 图1B ）。在盖子上切割一个15厘米直径的孔（ 图1C ）。 </li><li>将尼龙网切成25×25厘米的正方形。 </li><li>将网格放在容器的顶部边缘上，然后...

<ol>
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A., 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=Stringent+analysis+of+gene+function+and+protein-protein+interactions+using+fluorescently+tagged+genes.">Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.</a> Genetics. 190 (3), 931-940 (2012).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev Dyn. 232 (3), 827-834 (2005).</li><li>Mukherjee, A., 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=Regulation+of+Notch+signalling+by+non-visual+beta-arrestin.">Regulation of Notch signalling by non-visual beta-arrestin.</a> Nat Cell Biol. 7 (12), 1191-1201 (2005).</li><li>Gilbert, M. 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L., 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=Riquiqui+and+minibrain+are+regulators+of+the+hippo+pathway+downstream+of+Dachsous.">Riquiqui and minibrain are regulators of the hippo pathway downstream of Dachsous.</a> Nat Cell Biol. 15 (10), 1176-1185 (2013).</li><li>Zhang, 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=The+ecdysone+receptor+coactivator+Taiman+links+Yorkie+to+transcriptional+control+of+germline+stem+cell+factors+in+somatic+tissue.">The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue.</a> Dev Cell. 34 (2), 168-180 (2015).</li><li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+by+receptor+tyrosine+kinases.">Cell signaling by receptor tyrosine kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Vincent, J. P., Girdham, C. H., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-autonomous,+ubiquitous+marker+for+the+analysis+of+Drosophila+genetic+mosaics.">A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics.</a> Dev Biol. 164 (1), 328-331 (1994).</li><li>Zhai, B., Villen, J., Beausoleil, S. A., Mintseris, J., Gygi, S. 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H., Peng, J., Mayor, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+isolate+ubiquitin+conjugates+reveals+wide+role+for+ubiquitination+during+neural+development.">A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.</a> Mol Cell Proteomics. 10 (5), (2011).</li><li>Ramirez, J., 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=Proteomic+Analysis+of+the+Ubiquitin+Landscape+in+the+Drosophila+Embryonic+Nervous+System+and+the+Adult+Photoreceptor+Cells.">Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.</a> PLoS One. 10 (10), 0139083 (2015).</li></ol>

这里提出的方案是一个简单和一般的程序，用于在中等规模设置果蝇种群笼子，并从胚胎中制备全细胞蛋白质提取物。所得提取物可用于各种下游应用，例如在亲和树脂上纯化蛋白质复合物。在冰上进行提取步骤和使用强烈的蛋白酶抑制是至关重要的，以最小化蛋白质降解。如所述，该方案适用于分离大多数细胞质和一些膜和可溶性核蛋白6,10,12,15。它可以容易地适应于来自其他隔室的更集...

遗传筛选和最近的基因组方法已经彻底改变了生物功能的研究。然而，重要的细胞信息被编码在蛋白质及其相互作用的合作伙伴的整体中。虽然传统的基因修饰屏可以识别速率限制途径成分并恢复间接相互作用，但蛋白质组学方法的强度在于它们识别感兴趣的蛋白质的完全即时相互作用网络的能力。因此，蛋白质组学是研究生物系统的有价值的正交方法，补充基因组学，转录组学和传统遗传筛选。亲和纯化 - 质谱（AP-MS）已被证明是研究细胞和组织中天然环境中蛋白质 - 蛋白质相互作用（PPI）的有力方法。这种方法允许在特定发展中识别直接或间接的相互作用已经成功地用于鉴定多种发育途径中的多种新型PPI（参考文献1 ）。尽管PPI研究取得了无可争议的成功，但其大部分已经在培养的细胞中进行，其中感兴趣的“诱饵”蛋白质被过度表达。在细胞培养中研究PPI有两个问题：首先，特定的细胞系由于缺乏某些蛋白质的表达而不能提供完整的相互作用。第二，通常在这种分析中使用的高过表达可能导致人为的物质，如蛋白质错误折叠或假阳性相互作用的识别。 这两个限制都可以通过在体内分析PPI来克服。这种实验中的限制性步骤是可用于纯化蛋白质复合物的原料。 黑腹果蝇长期以来一直被用作功能分析的模型sis，最近也被证明是在体内研究PPIs的优秀体系。 果蝇胚胎发生是研究PPI的特别有吸引力的组织类型，因为可以容易地收集胚胎，并且也因为大多数基因（&gt; 88％）在胚胎发生过程中被表达，因此提供了丰富的体内环境来检测相关的PPIs 3 。 传统上，苍蝇的生物化学研究利用非常大规模的胚胎收集（100-150 g），如纯化功能转录裂解物4,5所必需的。以前在果蝇中的 AP-MS研究也需要大量的胚胎（ 5-10g ），因为它们依赖于两步纯化方法，例如串联亲和纯化（TAP），每个步骤6都有相关的材料损失。大型的起始材料需要在大型人口笼中设置胚胎收集，这可能是昂贵的（在商业购买时）并且耗时地维持和清洁7,8,9 。 单步亲和纯化方法的发展的最新进展以及质谱仪的增加的灵敏度已将原料的必要量减少一个数量级。使用诸如链霉抗生物素蛋白结合肽（SBP）或绿色荧光蛋白（GFP）的标签并从小于1g的胚胎开始，可以分离足以鉴定的诱饵蛋白质和相互作用成分的量质谱法10,11 。 这里提出的协议的目标是帮助研究人员我感受到体内 PPI 的生物化学分析的障碍。为此，我们提供了一种简单而廉价的方法来收集中等规模（0.5-1 g）的果蝇胚胎，然后一步制备适合随后通过AP-MS或其他方法分析的全细胞蛋白质提取物。我们的方法依赖于使用可由任何实验室轻松生产的定制的1-L或5-L种群笼。此外，这里提出的提取条件已经在几个研究中验证，在培养的细胞中和在体内 10,12,13,14,15,16,17。

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Container (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209330</td>
			<td style="width:644px;">For making 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Lid (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209320</td>
			<td style="width:644px;">Diameter 8.37 in. Lids for 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 100 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 1-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 150 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875714</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 60 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:644px;">Material: Polystyrene. Used during embryo dechorionation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sefar NITEX nylon mesh</td>
			<td style="width:183px;">Sefar</td>
			<td style="width:119px;">03-180/44</td>
			<td style="width:644px;">180 micron, 44% open area, used for fly cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Milwaukee 3 in. Hole Dozer Hole Saw with Arbor</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">49-56-9670</td>
			<td style="width:644px;">3 inch cutting tool for making 1-L cages.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Wide-Mouth Straight-Sided PMP Jars with White Polypropylene Screw Closure</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">11-823-33</td>
			<td style="width:644px;">For making 1-L cages. Thermo Scientific cat # 21171000. Alternative 1-L containers can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Red Star Active Dry Yeast, 2 pound pouch</td>
			<td style="width:183px;">Red Star&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from Amazon.com or other suppliers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Frozen 100% Apple Juice Concentrate&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from a grocery store. Has to say &#34;100% apple juice&#34; on the can.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:183px;">Acros Organics</td>
			<td style="width:119px;">AC126965000</td>
			<td style="width:644px;">also known as methylparaben or Tegosept. Used as a preservative in AJ plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">IGEPAL CA-630 for molecular biology, 100 ml</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">I8896</td>
			<td style="width:644px;">used for preparing lysis buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Rapid-Flow Sterile Disposable Filter Units with CN Membrane&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-740-2A</td>
			<td style="width:644px;">0.2 &#956;m pore size. Used for filtering 5x lysis buffer. Thermo Scientific cat # 1260020.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">CO-RO ROCHE cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">11697498001</td>
			<td style="width:644px;">Vial of 20 tablets. Used for protease inhibition in lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning SFCA syringe filters&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-754-21</td>
			<td style="width:644px;">SFCA membrane, diameter 26 mm, pore size 0.45 &#956;m. Used for filtering final extract samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">BD&#160;Luer-Lok Disposable Syringes without Needles</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">14-823-2A</td>
			<td style="width:644px;">for filtering final extract samples. BD cat # 309604.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:439px;">Bleach</td>
			<td style="width:183px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">used for embryo dechorionation at 50% (vol/vol) in water, can be purchased at any supermarket. It is important to use the Clorox brand, as other brands may result in incomplete dechorionation or may be toxic for embryos.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning Costar Netwell Plates</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-213</td>
			<td style="width:644px;">mesh containers for embryo collections. 74 &#956;m mesh size, 6-well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Wheaton Dounce Tissue Grinders, capacity 15 ml</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">06-435B</td>
			<td style="width:644px;">homogenizer for embryos, with loose and tight pestles. Wheaton cat # 357544.</td>
		</tr>
	</tbody>
</table>

medium-scale preparation of drosophila embryo extracts for proteomic experiments

蛋白质 - 蛋白质相互作用（PPI）的分析已经成为研究生物过程和机制，如细胞信号传导，生物发育和疾病的不可或缺的方法。通常希望使用体内材料获得PPI信息，以获得最自然和无偏见的互动网络视图。果蝇果蝇黑腹果蝇是体内研究PPIs的绝佳平台，适用于生物化学实验隔离材料的简单方法。特别地，由于在该发育阶段易于收集动物以及大多数蛋白质在胚胎发生中表达，所以果蝇胚代表研究PPI的便利类型的组织，从而提供了显示大多数PPI的相关环境。在这里，我们提出了中等规模收集果蝇胚胎的方案（0.5-1 g），这是一个广泛的蛋白质组学应用，包括通过亲和纯化 - 质谱（AP-MS）分析PPI。我们描述我们的设计为1 L和5 L网箱的胚胎收集，可以轻松，廉价地设置在任何实验室。我们还提供用于胚胎收集和蛋白质提取的通用方案，以产生可直接用于下游应用如AP-MS的裂解物。我们的目标是为所有研究人员提供一种可行的手段来进行体内 PPI的分析。

为了说明在蛋白质复合物纯化实验中使用该方案，我们在控制下产生了一种纯合可行的飞行线，表达EGFP标记的果蝇细胞外信号调节激酶（由滚动基因编码的ERK）的臂EGFP-ERK的普遍存在的犰狳 （ 臂 ）启动子18,19 。在果蝇胚胎发生的大多数阶段， 臂启动子是有活性的。使用所述方案制备裂解物，通过蛋白质印...

Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

该方案的目标是提供一种直接和廉价的方法来收集中等规模的果蝇胚胎（0.5-1 g），并制备可用于下游蛋白质组学应用的蛋白质提取物，如亲和纯化 - 质谱（AP-MS ）。

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Research

JoVE Journal

Developmental Biology

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

科研

发育生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...