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，然后用盖子向下按压（ 图1C ）。确保网格紧固，盖子不会倾斜。笼子已准备好安装苍蝇（参见步骤5.3）。 </li></ol> 2.制备1L飞行笼（适合10cm板） <ol><li>获得所需材料:电钻，3英寸（76毫米）切割工具（ 图1E和F）图1F​​），带盖的直立1L塑料罐，尼龙网，剃刀刀片，砂纸，护目镜和工作手套。 </li><li>在底部和塑料瓶的盖子上切下3英寸（76毫米）的孔（ 图1G ），盖子在钻孔时紧紧地拧入容器。开始制作一个孔时，将切削刀具定位在圆心的中央。 注意:在此步骤中佩戴眼睛防护和工作手套。 </li><li>用剃刀刀片和砂纸平滑孔的边缘。 </li><li>将尼龙网切成18×18厘米的正方形。 </li><li>将网格放在瓶子的顶部边缘上并拧紧盖子（ 图1G ）。确保网格紧固，盖子不会倾斜。笼子已准备好安装苍蝇（参见步骤5.3）。 </li></ol> 3.苹果汁（AJ）板的制备<ol><li>将19g琼脂和1L18.2MΩ·cm的水加入2L烧瓶中。添加搅拌棒，并简单混合。 </li><li>高压灭菌30分钟同时，从冷冻原料中解冻100％苹果汁浓缩物，并制备10mL 10％（重量/体积）4-羟基苯甲酸甲酯溶液在乙醇中。 </li><li>高压灭菌后，开始将搅拌板上的琼脂混合，加入80 mL 100％苹果汁浓缩液和10mL 10％（重量/体积）的4-羟基苯甲酸甲酯溶液至烧瓶中。在室温下保持恒温混合。 </li><li>倒入15厘米培养皿（5升笼子）或10厘米培养皿（1升笼子），以使每个培养皿中的琼脂水平约为5毫米厚。允许在室温下固化，并包装在塑料袋中。 AJ板可以在4℃下储存两周。 注意:培养皿可以洗涤和重复使用。 </li><li>准备湿酵母糊:将一些活性干酵母倒入具有容易混合的盖子的100mL容器中，并在搅拌下少量加水，得到浓稠但充分混合糊。在4°C下储存长达2周。 </li></ol> 4.裂解试剂的制备<ol><li> 制备5x裂解缓冲液:250mM Tris pH 7.5,25％甘油，1％辛基苯氧基聚（乙烯氧基）乙醇（IGEPAL CA-630），7.5mM MgCl 2，625mM NaCl，125mM NaF，5mM Na 3 VO 4 。 <ol><li>制成200毫升:将1g NaF粉末和184毫克Na 3 VO 4粉末在不断搅拌下在71毫升水中完全溶解。加入50mL 1M Tris pH 7.5,2mL 100％IGEPAL，1.5mL 1M MgCl 2和25mL 5M NaCl。然后加入50mL甘油（最后加入），继续搅拌1小时。 </li><li>使用250 mL过滤器过滤灭菌。该步骤可能很慢，但过滤溶液对于去除不溶性颗粒很重要。在50mL管中分装10 mL，储存于-80°C。 </li></ol></li><li>在水中制备1 M二硫苏糖醇（DTT）溶液。在-20℃下以1mL等分试样储存。 </li><li>获得蛋白质ase抑制剂混合片，与乙二胺四乙酸（EDTA）。储存于4°C。 </li><li>获得10 mL注射器和注射器过滤器:直径26 mm，无表面活性剂的醋酸纤维素（SFCA）膜，孔径0.45μm。 </li></ol>飞鸟收集<ol><li>扩增携带有标签的蛋白质的转基因飞线（每瓶约100只苍蝇），每2天转运瓶子，直到每条飞行线累积25-30瓶。以类似的方式放大控制库存（ 例如 yw线）。 </li><li>温热AJ板至室温（约1-2小时，可以一夜之间完成）。使用戴手套的手指，将一些湿酵母膏撒在AJ板的中心， 6厘米圆形，15厘米板和4厘米圆形的10厘米板。 </li><li> 使用标准的果蝇阶段设置，用二氧化碳气流麻醉苍蝇，并转入笼子（每5L笼子大约15只成熟的苍蝇的瓶子，每1L笼子5瓶）。 <ol><li>用步骤5.2制备的AJ板覆盖笼底，并使用两条胶带将AJ板粘贴到笼上（ 图1D和1H ）。折叠板上的胶带的顶端，以便在更换印版时易于剥离是有用的。 </li><li>苍蝇从麻醉中恢复后，将笼子保持在底部，并在顶部网格。 注意:重要的是等到苍蝇完全恢复，否则它们会粘在AJ板和酵母酱上。 </li><li>在实验结束时，为了准备笼子进行清洁，将笼子放入冷藏室几个小时。将网状物浸泡在水中几个小时将有助于清洁。 </li></ol></li><li>孵化笼中的苍蝇在室温2-3天，并更换AJ板每天两次。要更换盘子，请将笼子倒置，从盘子上取出胶带，然后将盘子固定在笼子上，轻轻地将整个笼子轻轻地放在板凳上，快速地将旧板子换掉一个新的盘子，尽量不要让苍蝇逃跑。 注意:苍蝇需要适应笼子，最好从第3天开始。他们将在高峰期继续铺设约4-5天。两个5 L的笼养能力可以在过夜收集中产生高达1 g的胚胎。使用1 L笼子进行较小规模的实验。 </li><li>允许苍蝇将新鲜AJ板上的胚胎过夜（约16小时）或任何其他所需的时间长度。 </li><li>第二天早上，将每个飞行线用于每个飞行线，用于胚胎收集的标记和称重网格容器。 </li><li> 使用第4部分的试剂制备1x裂解缓冲液（注:即将使用的1x裂解缓冲液必须在提取前准备）:向10 mL 5x浓缩的裂解缓冲液（储存于-80°C）中加入40 mL水，在50mL管中。 <ol><li>加入50μL的1 MDTT至终浓度为1mM，充分混合并分成两个50mL管，每个管中25mL。向其中一个管中加入一种蛋白酶抑制剂片剂，并在4℃下旋转30分钟。当片剂溶解，收集和解冻胚胎（步骤5.8至6.6）。检查以确保平板电脑已完全溶解，并将缓冲液保持在冰上。 注意:带有DTT的1x缓冲液管可以保存在-80°C，只需要在下一次实验之前添加平板电脑。考虑到蛋白质纯化步骤中的后续洗涤，25mL裂解缓冲液对于多达2个样品是足够的。 </li></ol></li><li>将AJ板放在使用飞线的基因型的笼子上，并将盘更换为新鲜的。 注意:在此步骤中可以从板上刮掉过量的酵母膏。胚胎可以在25℃或室温下在板上老化，以获得期望的发育时间窗口。 </li><li> 添加足够的水to覆盖每个AJ板。用柔软的油漆刷轻轻地将胚胎从板上取下（平头约10毫米宽适用于大板）。将胚胎倒入以前称重和标记的网状容器中，并排出多余的水分。 <ol><li>如果需要，进行第二次清洗和刷涂板以除去所有的胚胎。将来自相同飞线的附加板的胚胎合并到相同的网状容器中。 </li></ol></li><li>简单地在纸巾上清除网眼以除去多余的水分。 </li></ol>胚胎脱层<ol><li>半满6厘米培养皿与50％漂白剂（体积/体积溶液在水中）。 注意:请参见材料和试剂表中关于漂白剂品牌的重要评论。 注意:戴上手套，避免在衣服上漂白。 </li><li>准备2个较大的培养皿用水。 </li><li>将含有胚胎的网状容器浸入50％漂白剂中90秒。在入口处轻轻点击网格在漂白剂溶液中暂停胚胎几次。 </li><li>在90s孵育结束时，用水洗涤每个2个皿中的网状容器约10s。 </li><li>用喷水瓶用18.2MΩ·cm的水冲洗网状容器。此外，洗涤网状容器的侧面和外部。 注意:彻底清洗后不应检测到漂白气味。 </li><li>将网状容器放在纸巾上。用胚胎称重网状容器，并减去步骤5.6中获得的空容器的重量。记录胚胎的最终重量。适量的胚胎是0.5 - 1 g。 </li></ol> 7.胚胎提取物的制备<ol><li>加入1 mL具有蛋白酶抑制剂的裂解缓冲液（从步骤5.7）至均匀玻璃匀浆器，并保持在冰上。将胚胎从网状容器转移到匀浆器中。 注意:胚胎可以用平头o拾取金属刮刀或刷子直接转移到缓冲器中。在冰面上执行本节中的所有步骤。 </li><li>用额外的1 mL裂解缓冲液洗涤网上剩余的胚胎，并加入匀浆器中的材料。让胚胎沉淀到匀浆器的底部，并小心吸出上清液。 </li><li>将蛋白酶抑制剂（来自步骤5.7）的裂解缓冲液加入匀浆器中，目标为每克胚胎5-6mL裂解缓冲液。 </li><li>用松散的杵将胚胎均匀化4-6次。 注意:在第一次冲击中，会有更多的阻力。尝试连续移动杵，以免溅出溶液。 </li><li>用紧密的杵将胚胎均匀化8-10次。 </li><li>将匀浆在冰上孵育20分钟。 </li><li>将匀浆转移到微量离心管中，并以最高速度（约13,000 xg）在4℃离心15分钟。 </li><li> Avoidi将颗粒和最上层脂质层转移到新鲜的微量离心管中，并以最高速度（约13,000xg）在4℃离心15分钟。 </li><li>仔细吸取1毫升尖端的上清液，并装入冷冻的10mL注射器中，并连接0.45μm过滤器（将相同样品的等分试样装入一个注射器）。将所有溶液推入冰上标记的15 mL管中。提取物可以立即用于随后的蛋白质纯化实验或在液氮中快速冷冻，并保持在-80℃以备将来使用。 注意:最好将提取物而不是胚胎冷冻，因为在随后的解冻过程中，蛋白酶可能会被激活并导致蛋白质降解。 </li></ol>

<ol>
	<li>Veraksa, A. <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+developmental+processes:+insights+from+mass+spectrometry-based+proteomics.">Regulation of developmental processes: insights from mass spectrometry-based proteomics.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 723-734 (2013).</li><li>Gavin, A. C., Maeda, K., Kuhner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+charting+protein-protein+interaction:+mass+spectrometry-based+approaches.">Recent advances in charting protein-protein interaction: mass spectrometry-based approaches.</a> Curr Opin Biotechnol. 22 (1), 42-49 (2011).</li><li>Arbeitman, M. N., 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=Gene+expression+during+the+life+cycle+of+Drosophila+melanogaster.">Gene expression during the life cycle of Drosophila melanogaster.</a> Science. 297 (5590), 2270-2275 (2002).</li><li>Soeller, W. C., Poole, S. J., Kornberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcription+of+the+Drosophila+engrailed+gene.">In vitro transcription of the Drosophila engrailed gene.</a> Genes Dev. 2 (1), 68-81 (1988).</li><li>Kamakaka, R. T., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soluble+nuclear+fraction,+a+highly+efficient+transcription+extract+from+Drosophila+embryos.">The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos.</a> Methods Cell Biol. 44, 225-235 (1994).</li><li>Kyriakakis, P., Tipping, M., Abed, L., Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+in+Drosophila:+The+advantages+of+the+GS-TAP+system.">Tandem affinity purification in Drosophila: The advantages of the GS-TAP system.</a> Fly (Austin). 2 (4), 229-235 (2008).</li><li>Shaffer, C. D., Wuller, J. M., Elgin, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raising+large+quantities+of+Drosophila+for+biochemical+experiments.">Raising large quantities of Drosophila for biochemical experiments.</a> Methods Cell Biol. 44, 99-108 (1994).</li><li>Sisson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+large+populations+of+Drosophila+for+protein+biochemistry.">Culturing large populations of Drosophila for protein biochemistry.</a> CSH Protoc. 2007, (2007).</li><li>Caravaca, J. M., Lei, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+a+Drosophila+melanogaster+Population+Cage.">Maintenance of a Drosophila melanogaster Population Cage.</a> J Vis Exp. (109), (2016).</li><li>Yang, 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=Minibrain+and+Wings+apart+control+organ+growth+and+tissue+patterning+through+down-regulation+of+Capicua.">Minibrain and Wings apart control organ growth and tissue patterning through down-regulation of Capicua.</a> Proc Natl Acad Sci U S A. 113 (38), 10583-10588 (2016).</li><li>Neumuller, R. 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. M., Tipping, M., Veraksa, A., Moberg, K. 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+Screen+for+Conditional+Growth+Suppressor+Genes+Identifies+the+Drosophila+Homolog+of+HD-PTP+as+a+Regulator+of+the+Oncoprotein+Yorkie.">A Screen for Conditional Growth Suppressor Genes Identifies the Drosophila Homolog of HD-PTP as a Regulator of the Oncoprotein Yorkie.</a> Dev Cell. 20 (5), 700-712 (2011).</li><li>Anjum, S. G., 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+Toll+Signaling+and+Inflammation+by+beta-Arrestin+and+the+SUMO+Protease+Ulp1.">Regulation of Toll Signaling and Inflammation by beta-Arrestin and the SUMO Protease Ulp1.</a> Genetics. 195 (4), 1307-1317 (2013).</li><li>Degoutin, J. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+Drosophila+melanogaster+embryos.">Phosphoproteome analysis of Drosophila melanogaster embryos.</a> J Proteome Res. 7 (4), 1675-1682 (2008).</li><li>Franco, M., Seyfried, N. T., Brand, A. 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>

作者没有什么可以披露的。

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

medium-scale-preparation-drosophila-embryo-extracts-for-proteomic

Research

JoVE Journal

Developmental Biology

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

7.8K 次观看.  University of Massachusetts Boston. 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 ）。 

视频: Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

科研

遗传学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

生物学

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

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

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

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

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 ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

发育生物学

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Here, a protocol for the implementation of expansion microscopy in early Drosophila embryos to achieve super-resolution imaging using a conventional laser-scanning confocal microscope is presented.

使用扩增显微镜物理放大 整个果蝇 胚胎以进行超分辨率成像

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged ...

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

中等规模的制备

摘要

探索更多视频

此视频中的章节

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

研究有丝分裂的显微注射技术果蝇合胞体胚胎

电子显微镜对胚胎的制备果蝇胚胎心管

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

使用扩增显微镜物理放大整个果蝇胚胎以进行超分辨率成像

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

胚胎运动神经元轴突投影模式的可视化

中等规模的制备

摘要

探索更多视频

此视频中的章节

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

研究有丝分裂的显微注射技术果蝇合胞体胚胎

电子显微镜对胚胎的制备果蝇胚胎心管

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

使用扩增显微镜物理放大 整个果蝇 胚胎以进行超分辨率成像

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

胚胎运动神经元轴突投影模式的可视化

使用扩增显微镜物理放大整个果蝇胚胎以进行超分辨率成像