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，转移混合物B到A。混合轻轻保持搅拌组合的混合物，让它冷却15分钟。避免过度冷却，并加入防腐剂之前所得到琼脂凝固。 </li><li>使10％（重量/体积）甲基乙醇4 - 羟基苯甲酸溶液。这将作为防腐剂。加3.75毫升解决上述苹果汁琼脂混合物。继续搅拌anothe令第5分钟。 </li><li>倒125毫升苹果汁琼脂混合物，防腐剂为各300 平方厘米的塑料或泡沫塑料板。让它冷却至室温，并巩固。避免气泡的形成，同时浇筑。 注：苹果汁琼脂平板上就可以立即使用。覆盖未使用的板材有明确的包裹，并在4℃下保存长达一个星期。 </li></ol> 2， 果蝇胚胎收集和预交联处理<ol><li>加载人口笼约40,000至50,000苍蝇从胚胎将被收集。 </li><li>使每片含125毫升标准玉米面，糖蜜培养基12两个板块，洒上约4g每盘干面包酵母。将这些板块进笼子人口在​​25℃培养箱中培养48小时催肥的苍蝇。 </li><li>暖2苹果汁琼脂平板上25℃。撒上约1g每个干面包酵母板。将2板块在人口笼开始收集0-1小时大的胚胎。 </li><li>取出板在1小时收集的末端。有2个新板更换是否需要新一轮的集合。 </li><li>加入足够的水覆盖板和用细画笔轻轻悬浮胚胎。通过做出来的不锈钢丝网两层筛子倒入悬浮胚胎。 注：顶层是由中等孔径（850微米）目，同时通过让胚胎用于收集大飞零部件。底部层由细网孔（75微米），而通过酵母和水让其收集胚胎。 </li><li>用优良的油漆刷将采集到的胚胎转移到一个50毫升管和细尼龙网制成的集合篮子。用清水冲洗短暂的胚胎，然后涂抹在纸巾网格晾干。 </li><li>沉浸胚胎在50％漂白伴以轻轻旋转3分钟。彻底冲洗LY与水以除去任何残留的漂白剂。涂抹在纸巾网格晾干。 </li><li>沉浸在异丙醇，轻轻摇动15秒或直到多胚团块破裂的dechorionated胚胎（这不应该超过30秒）。涂抹在纸巾网格彻底干燥。 </li><li>而用吸管流庚烷在胚胎，让他们重悬沉浸在庚烷5分钟的胚胎。涂抹在纸巾网格晾干。 </li><li>冲洗的胚用磷酸盐缓冲盐水，pH值7.4（PBS）中的含有0.1％Triton X-100的PBS（PBST）3分钟流。 </li><li>拆卸的收集筐和沿着与网格转移胚胎到15毫升管含有10ml PBST。颠倒离心管轻轻洗胚脱网。 </li><li>从管中取出的网格。把该管处于垂直位置，并让胚由重力下沉到试管底部。 注：约100 - 300微升胚胎预期用于从一个轿厢的1小时收集。 </li><li>除去PBST，继续执行立即交联。 </li></ol> 3， 在体内收集胚胎的交联<ol><li>在使用前配制0.2％的甲醛在PBST。加10毫升甲醛固定液的胚胎孵育在25℃下用旋转振荡器上10分钟，温和搅拌。由一天结束时丢弃未使用的固定剂。 </li><li>淬灭交联反应<ol><li>在交联结束时，让胚胎沉到试管底部。 </li><li>除去甲醛溶液。立即加入10毫升的0.25M甘氨酸在PBST中以淬灭交联反应。孵育在25℃下用旋转振荡器上5分钟，温和搅拌。 </li><li>让胚胎沉到试管底部。取出淬火液。用10ml PBST中的3次洗涤胚胎。 </li><li>彻底删除对P用移液管BST洗涤溶液。店铺胚胎在-80℃直至使用。 </li></ol></li></ol> 4，蛋白样品制备和免疫<ol><li>在2毫升裂解缓冲液用Dounce匀浆器匀化500μl的交联的胚胎（0.5M尿素，0.01％SDS，2％的Triton X-100，2mM的苯甲基磺酰氟[PMSF]和蛋白酶抑制剂混合物的PBS中）。 注：除非另有规定执行这一点，下面的步骤在室温。 </li><li>裂解物转移到1.5毫升离心管中，轻轻地摇动15分钟，在旋转平台上，以确保彻底溶解。离心溶胞产物以16,000×g离心5分钟以沉淀细胞碎片。除上清液在一个干净的15ml试管中。 </li><li>加40微升抗-HA琼脂糖珠浆液上清液孵育2.5小时的旋转平台上。 </li><li>通过离心沉淀珠粒在2500×g离心5分钟。取出上清液，但离开欧普罗ximately250μl的在管内。重悬珠在剩余的上清液并转移到1.5ml的离心柱用10微米孔径的过滤器。 </li><li>离心旋转柱以12,000×g离心10秒，并通过丢弃的流动。 </li><li>通过添加200μl的洗涤缓冲液（0.1M甘氨酸，1M氯化钠，1％IGEPAL CA-630，0.1％Tween-20中，在PBS中）洗涤珠。离心柱以12,000×g离心10秒，弃去洗涤。重复上述洗涤2次以上。 </li><li>加40微升洗脱缓冲液（2毫克/毫升的HA-肽溶于PBS中）的小珠和在37℃下进行15分钟，温和搅拌每隔5分钟。离心柱在12000×g离心10秒，收集洗脱液。 </li><li>用抗HA抗体13确认由Western印迹分析复杂的洗脱。 </li></ol>

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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 ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

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

16.6K 次观看.  Murray State University. 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。 

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

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

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

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

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

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

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

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 ...

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

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

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

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

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

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

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

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

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

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

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

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

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

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

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 ...