我在此协议的发展感谢以下人员的协助：查克·克里斯腾森，葛丽泰·宾福德，亚历克斯·K斯特，康拉德Zinsmaier和梅斯伊马德。由NIEHS资助ES06694到SWEHSC，NIH / NCI资助CA023074到AZCC支持亚利桑那州蛋白质组学协会和亚利桑那大学的BIO5研究所被收购质谱和蛋白质组学数据。资助这项工作是由美国国立卫生研究院（全科医学研究所）赠款1F32GM83661-01和1R15GM097714-01杰西卡E.者套装提供。

1.制备的电刺激的装置<ol><li>连接电刺激器电源线，插座和连接外部脚踏板的外部信号输入。 注：电线必须妥善绝缘，裸露的金属提供电流应不被感动。戴乳胶或丁腈手套保护。连接电源线和连接电线时电刺激器的电源开关应处于OFF位置。 </li><li>连接正极，以红色输出端上的电刺激器（当极性开关处于正常模式）和负电极连接到黑色的输出端相连。注：每个电极导线应终止于一个鳄鱼夹。 </li><li>设置刺激到以下推荐的初始设置：电压= 7 V，频率=每秒1个脉冲，延迟= 0毫秒，持续时间= 200毫秒，双脉冲开关=正规，​​模式切换= OFF。 </li><li>电极通过连接鳄鱼夹voltme测试输出器正面和负面的测试线。开启刺激器电源开关，并提供电流脚踏板。 </li></ol> 2.制备固定化镊子等材料<ol><li>沾羽镊子一个叉成液体的塑料涂层，并等待四小时让涂料干燥。紧紧包住相对叉齿的尖端15毫米与棉缝纫线的单个层，以及固定用线通过捆扎结束于一个结。 注意：该线程用于吸收施加的盐溶液，以促进导电，而塑料涂层提供绝缘以阻止电流。 </li><li>砍钝的皮下注射针头的针尖锋利的剪刀，顺利开通用砂纸。连接针在真空垃圾收集器（ 例如 ，真空过滤烧瓶）经由管道。 注：针会吸走水分和反刍，将成为完成由刺激器电极产生的电路。针口万亩圣足够小，以不伤害蜘蛛，但足够大以真空溶液而不堵塞（ 如 ，25的G轨距×1个半，参见材料清单）。 </li><li>位置轻量级镊子水平在使用乙烯树脂被覆双叉钳固定到下视解剖显微镜的字段一个磁性底座或固定台装置的密闭位置时，在顶部钳涂塑叉和螺纹涂覆叉上底部。 注意：双叉钳被维持在通过夹具夹持固定位置到磁性底座（参见材料清单）。一个橡胶圈可以紧密地固定围绕轻量级钳子的齿尖，旁边的夹持器，以增加额外的压力，以保持轻量级钳子在周围一次引入的蜘蛛闭合位置。 </li><li>连接正极鳄鱼夹钳“底部叉线的上游和高度负鳄鱼夹钝金属真空针。 </li&gt; <li>测试电路的电流与电压表通过将正极引线上线和鳄鱼夹，并把负极导线上的真空钝针之间钳爪。按脚踏板，以确保检测到足够的电流，并且如果检测到足够的电流，除去引线。 </li><li>准备几个微细管毒液集合。拥有一个单独的微毛细管的一端用戴着手套的手，并在用无菌镊子金属的另一端，水平放置钳持月底在本生灯火焰。拉在与金属镊子远离火焰来创建一个细长尖端微细管，同时保持另一​​端在一个固定的位置。 注意：戴防护眼镜作为微毛细管容易破裂。 </li><li>在培养皿中搁置在安装油灰条制备微毛细管。检查在解剖显微镜下微细管端，并创建一个小的，倾斜的开口在拉出端灭菌金属镊子。 </li><li>啦贝尔可变数量的无菌0.5毫升管。加无菌去离子水，无菌管中（ 例如 ，5微升灭菌的去离子水）。靠近管并置于试管置于冰上。 </li><li>制备盐溶液在烧杯中，并填充第二个烧杯用高压灭菌水。 </li></ol>在镊子3.固定蜘蛛<ol><li>打开CO 2罐，并从封闭的塑料瓶收集蜘蛛传输使用长金属镊子（见材料清单）成CO 2室。 注：黑寡妇蜘蛛有危险的毒液;总是这个协议时戴上手套。 </li><li>保持蜘蛛在室内5-10分钟或直到麻醉，不再动时，轻轻地打了招呼，长镊子。用移液管以润湿线程上钳子用生理盐水溶液。 注：有关二氧化碳麻醉参数黑寡妇指南，请参阅西班牙广场和摩尔19。 </li><li>从CO 2检索麻醉蜘蛛室通过使用钝解剖钳和手套的手的腿前采摘它。移动麻醉蜘蛛轻量级钳装置。封闭轻量级镊子尖头分开，并把蜘蛛的头胸部的插脚之间，让叉子紧紧地固定住，但没有被压碎，收关，确保蜘蛛。确保蜘蛛放置甲壳背侧面向下（见Foelix 20，用于引导到蜘蛛解剖学），搁在螺纹涂覆叉，和甲壳的腹侧朝向塑料涂层，而正极仍然附着于底叉。蜘蛛处理工作时快。 </li><li>调整解剖显微镜的放大倍率，对焦和光源，直到蜘蛛的螯肢和獠牙都清晰可见。 </li></ol> 4.收集毒液<ol><li>打开真空并利用第二钝尖注射器Ñ喷蜘蛛螯肢溶液与水eedle。吸走水用真空针以除去杂质。 </li><li>触摸真空针附着负极蜘蛛主席台和提供脉冲与脚踏板的一个或更多次。如果有必要的真空客场吐出。 注：蜘蛛是主席台后口里面螯肢的螯肢和endites（见Foelix 20详细解剖蜘蛛）之间的背侧面。注意：对于每一个脉冲，蜘蛛的腿合同，毒液会显示为明显的水滴摆脱獠牙。 </li><li>收集毒液水滴与微细管尖和传输毛细管末端浸入0.5毫升管与水或缓冲溶液（溶液将被吸入到毛细管）。 注意：避免穿刺蜘蛛与毛细管可能导致血淋巴污染而死亡。此外，还要避免毛细血管的污染丝绸和胶水从喷丝头。 </li><li>连接传输注射器管适配器微毛细管（在端相对的收集尖）和dispense毒液液回管。把管上的冰。 注：在附近的锐器容器处置废物的毛细血管。 </li><li>将打开塑料瓶收集，伴随着它的上限，接近蜘蛛。小心地抓住蜘蛛的腿在一个手持钝解剖钳小心地打开羽量级镊子尖头用另一只手，滑蜘蛛进入收集瓶钝钳，迅速接近上限。继续在-80℃的冰柜旁边的蜘蛛和储存含毒液管时结束。 注：继续从钳子移动时，蛛放回瓶中谨慎操作。确保收集瓶和瓶盖就在附近，以蜘蛛为它的快速传递和戴手套。 </li></ol> 5.毒液腺解剖<ol><li>蜂毒采集后两到三天，麻醉蜘蛛在CO 2室（见步骤3.1）。填写液氮载体和地点旁边的解剖显微镜。 注：WH恩正与液态氮，用眼保护和低温手套。 </li><li>干净的夹层区和解剖钳与解决方案，消除RNA酶和DNA污染。填补小培养皿下解剖显微镜解剖缓冲液（ 如柠檬酸1X盐水钠（SSC）缓冲液）放置。 </li><li>从CO 2室转让蜘蛛培养皿中使用镊子，迅速 ​​从腹部分开的头胸部。 </li><li>握住解剖显微镜下用镊子将头胸部和螯肢定位到视野。使用第二个钳子的两端尖锐削减角质层加入甲壳的螯肢的横向方面。横向抓住用第二个钳子的螯肢，轻轻拉扯来回，直到毒腺被拉出。 </li><li>从螯肢单独的腺体（或离开附着螯肢如果需要的话），并转移到cyrosafe0.5毫升管。地方CLOS编管在液氮中。 注：每个夹层应不超过15分钟。 </li><li>直到完成重复清扫额外的蜘蛛。从液氮输送管到-80℃的冰箱中。 </li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+geographic+and+intersexual+chemical+variation+in+venoms+of+the+spider+Tegenaria+agrestis+(Agelenidae).">An analysis of geographic and intersexual chemical variation in venoms of the spider Tegenaria agrestis (Agelenidae).</a> Toxicon. 39, 955-968 (2001).</li><li>Alape-Gir&#243;n, 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=Snake+venomics+of+the+lancehead+pitviper+Bothrops+asper:+geographic,+individual,+and+ontogenetic+variations.">Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations.</a> J. Proteome. Res. 7 (8), 3556-3571 (2008).</li></ol>

笔者有没有透露，也没有竞争的财务权益。

毒液代表生理活性蛋白质，肽和其他分子与药物发现应用中的一个重要来源，以及用于蜂窝和生态研究1-3的基本方面。然而，毒液的收集，特别是从危险的或小动物，是一项具有挑战性的任务。此协议演示如何毒液和毒腺可以从黑寡妇蜘蛛被收集，并证实这种方法通过从毒液克 ​​隆毒液MuDPIT分析和从cDNA中衍生得到的蛋白质数据库的组合成功压盖21。虽然此协议行之有效的黑寡妇，中型蜘蛛，其他的毒液收集技术已经被采用但对于较大的mygalomorph（狼蛛一样）蜘蛛，如毒液的毒牙从进入玻璃吸管例如 ，24。后一种方法直接抽吸，不会对较小尺寸的蜘蛛未aggressi很好地工作已经。 此处所描述的毒液采集协议中的一个特别重要的方面是制备所述收集过程的优化的初始阶段，使得它变得更常规的，一致的和快捷。该协议最初是具有挑战性的掌握，但随着重复试验，变得更容易和更快。警告还敦促在涉及危险蜘蛛的处理，利用电流，细点玻璃微毛细管，注射器和针头的各个关键阶段。这是微整形毛细血管时穿戴适当的个人防护装备，如丁腈手套，实验室外套，长裤和封闭的鞋子，以及眼镜很重要。 另一个挑战性的方面来毒液的收集是通过任一蜘蛛，尤其Latrodectus物种，从该收集的量可以是里产生的少量毒液充其量mited每1-2个人微升。获得足够的毒液用于下游应用，如蛋白质凝胶或功能测定法可能需要来自多个个体毒液的组合成一个管。在这种情况下，毒液应该只从同一性别，个体发育阶段的个体组合，并且种群中给出一些毒液25,26识别间性，发育和地理变异。蜘蛛也可以表现出相当大的变化在毒液的个体，其中较少量可以反映压盖的最近消耗之间产生量。因此它可能是可取的最后进给后，收集毒液几天。如果小毒液被释放时，过大的电流不应该施加于蜘蛛，这可能导致角质层破裂，导致与血淋巴或死亡的毒液的污染。 毒液样本污染的蜘蛛SIlk组合或人为来源也应当通过使用无菌的或清洁的设备避免。尽管存在这些挑战，收集纯净的毒液，让蜘蛛活着，最好是获得来自毒液腺匀浆（不与其他细胞蛋白分离毒液成分），并杀死蜘蛛的方法。这也是非常重要的，以确保样品快速冷冻以防止蛋白质的降解。 毒液提取促进后续的毒液生产，从而刺激毒液基因表达的毒腺。因此，因为该协议允许蜘蛛毒液生存耗尽，其腺体可以解剖几天以后的点处的毒液的基因表达，预计足以用于基因研究，如转录克隆10,11（杀蜘蛛）。几个重要的注意事项，还必须采取毒腺解剖。重点应放在利用实验室设备，用品耳鼻喉科和试剂是免费的降解RNA核糖核酸酶的。因此，建议擦镊子及其它非一次性设备和表面与消除RNA酶和DNA污染的解决方案。的解剖应尽可能快地和直接冷冻，以进一步确保了组织的RNA的完整性。最后，解剖应该只对麻醉蜘蛛进行的，经过他们的头胸部和腹部迅速分开。 总之，这篇文章提供了一个验证协议来获取蜘蛛毒液与蛇毒腺。毒液和毒腺允许使用蛋白组学和转录组学方法的蛋白质和肽的组分的分离和表征。此外，毒液样本可表示的功能测定，其确定其构成分子的生物医学和药理学潜在的起点。几乎所有的蜘蛛毒液产生，并广泛潜水员个别物种合成毒液组分的sity表明毒液分子的广阔多样都尚未被发现13。因此，该协议提供了工具，调查存在于蜘蛛毒液的生物活性分子的丰富来源。

毒液的动物分泌物主要是注入到另一种动物的捕食或防卫的目的，同时也具有重要的生物学应用与生物医学相关1-3。只有某些动物合成的毒液，但它的生产是整个脊椎动物（ 如腔肠动物，锥形蜗牛，蝎子和蜘蛛）和脊椎动物（ 如蛇，一些鱼类和哺乳动物）分类学的广泛，因为它已经独立进化多次1,4 。毒液的生化特性典型地表明它们是由各种各样的蛋白质和肽，这在很大程度上作用于注射的动物的循环系统和中枢神经系统，以快速提供构成毒素1。有毒动物的一小部分可能对人体无害，与住区（synanthropic）分布的5种特别的威胁。毒液成分和功能活动的研究中发挥了重要作用脊椎动物细胞成分（特别是神经元离子通道）6和阐明的基本细胞过程（ 如神经分泌）7功能特性。此外，选择蛇毒肽在生物环境中非常有用的特性，包括其用途作为癌症疼痛治疗8,9和开采的候选药物先导和抗毒血清的发展。毒液在生态和进化研究1,4,10,11也发挥显着的作用，因此这些有害分泌物的收集有许多实用工具。 有&gt;蜘蛛（蜘蛛目顺序）40000中描述的物种，以及所有，但100多户蜘蛛藏有一对毒腺，在12獠牙终止之一。蜘蛛的高物种多样性表明，它们代表有毒生物的最大分支。然而，蜘蛛毒液的生化特性有升argely集中于少数最经常与人类的蛇毒素相关的物种。最近的蛋白质组学和转录组蜘蛛毒液的研究表明，它们通常含有许多独特的蛋白质和多肽2,13,14。在高通量的cDNA测序和质谱肽指纹图谱的进步极大地促进了这些毒液蛋白15的发现。然而，这样的工作开始的足够的毒液和/或从蜘蛛毒液腺体，以及详细的文档集合为这些技术很少16。 本文提出了一种协议，用于从黑寡妇蜘蛛毒液与蛇毒腺的集合，它可以适用于同样大小的蜘蛛。毒液中分离的腺体的集合使得识别被分泌到毒液，而不是执行其它细胞功能的蛋白质的蛋白质。黑寡妇和其它 L.atrodectus种被广泛认为是最危险的蜘蛛之一，由于其高度神经毒素，导致剧烈疼痛的人，有时伴有大量出汗，肌肉收缩，高血压，呼吸困难和斑片状瘫痪5。这里介绍的蜂毒采集协议使用电刺激来提供电流给麻醉蜘蛛引起肌肉收缩和毒液释放。毒液液滴被迅速收集用微毛细管和分装到管中冷冻储存。由于该协议涉及到有害程序，它只能由训练有素的个人表现和谨慎，请致电关键步骤。所收集的毒液具有许多用途，例如表征与构成分子2，对于生理实验或功能测定18的隔离，并刺激毒液基因的表达11。该协议结束了DESCR其表达的毒腺解剖和保存用于毒液特异性基因的克隆有用iption，已经显示出现2-3天之后在不同的蜘蛛毒液10,11耗尽。

<table border="0" cellpadding="0" cellspacing="0" width="1090">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Nerve and muscle stimulator (electro-stimulator)&#160;</td>
			<td style="width:153px;">Grass Technologies</td>
			<td style="width:107px;">SD9</td>
			<td style="width:435px;">http://www.grasstechnologies.com/products/stimulators/stimsd9.html</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Voltmeter</td>
			<td style="width:153px;">RadioShack</td>
			<td>22-223</td>
			<td>any generic voltmeter/multimeter can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pointed Featherweight Forceps</td>
			<td style="width:153px;">Bioquip</td>
			<td>4748</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasti Dip</td>
			<td style="width:153px;">Performix</td>
			<td></td>
			<td>Available at Ace Hardware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 G X 1 1/2in Precision Glide hypodermic syringe needle</td>
			<td style="width:153px;">BD Medical&#160;</td>
			<td>305190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum filter flask 1L</td>
			<td style="width:153px;">Nalgene</td>
			<td>DS4101-1000</td>
			<td>smaller flask sizes may also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buchner Two Piece funnel, 90 mm</td>
			<td style="width:153px;">Nalgene</td>
			<td style="width:107px;">4280-0900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 microlitter Capillary Bores (Micro capillaries)</td>
			<td style="width:153px;">VWR</td>
			<td>53508-375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting putty strip&#160;</td>
			<td style="width:153px;">Loctite</td>
			<td></td>
			<td>Available at Ace Hardware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand* General-Purpose Extra-Long Forceps Length: 11-13/16 in.</td>
			<td style="width:153px;">Fisher</td>
			<td>10-316C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSC Buffer, 20X (pH 7.0), Molecular Grade</td>
			<td style="width:153px;">Promega</td>
			<td>V4261</td>
			<td>can be made from stock chemicals (150 mM NaCl, 15 mM sodium citrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf safe-lock tubes 0.5 mL tubes (cryogenic safe)</td>
			<td style="width:153px;">Eppendorf</td>
			<td>22363611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene Dewar, 1L, HDPE, Liquid nitrogen benchtop flask</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>4150-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rnase Away</td>
			<td style="width:153px;">VWR</td>
			<td>53225-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Tweezers (dissecting forceps)</td>
			<td>EMS</td>
			<td>78310-0</td>
			<td>similar high-quality fine point forceps can be substituted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Foot switch/pedal</td>
			<td style="width:153px;">Linemaster Switch Corp.</td>
			<td style="width:107px;">491-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Two-pronged extension clamp, with vinyl covered sleeves, 8.5 inches</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">21570-007</td>
			<td>similar models could be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Clamp Holder</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">89084-746</td>
			<td>similar models could be substituted, must accommodate diameter of extension clamp rod</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Magnetic base (holds extension clamp via clamp holder)</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">300042-270</td>
			<td>similar apparatus able to securely hold extension clamp in fixed position may be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Plastic Collecting Vials (large/40 dram)</td>
			<td style="width:153px;">Bioquip</td>
			<td style="width:107px;">8940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cotton sewing thread</td>
			<td style="width:153px;">Threadart.com</td>
			<td style="width:107px;">&#160;THRCOT9</td>
			<td>similar product could be substituted</td>
		</tr>
	</tbody>
</table>

extraction of venom and venom gland microdissections from spiders for proteomic and transcriptomic analyses

毒液是化学上复杂的分泌物通常包含大量的蛋白质和肽具有不同的生理活性。蛇毒蛋白的功能特性具有重要的生物医学应用，包括药物引线或探针，用于细胞受体的识别。蜘蛛是有毒的有机物的大部分种类丰富分化体，但只有少数物种的毒液是公知的，这部分是由于与收集微量毒液从小型动物相关联的难度。本文提出了一种协议，用于毒液使用电刺激蜘蛛的采集，显示出对西方的黑寡妇（ 黑寡妇蜘蛛 ）的程序。收集的毒液是不同的下游分析，包括通过质谱直接鉴定蛋白质，功能分析，并为转录组研究的毒液基因表达的刺激是有用的。该技术具有如下优点：在该协议异迟毒液从整个腺匀浆，它不来自未分泌的毒液的一部分细胞蛋白分离真正毒液组分。代表性的结果表明，从所收集的样品用质谱已知毒液的肽的检测。毒液采集过程之后是一个协议，用于解剖蜘蛛毒液腺体，其结果表明这导致毒液表达的蛋白质和肽在序列水平上表征。

经常进行的毒液和毒腺解剖采集的序列水平10,11,15表征毒液蛋白质和多肽。收集的毒液，也可以使用在生理测定以确定它们的功能活动18。毒液的收集将刺激毒腺基因的表达，从而促进特定毒素的成绩单，通过RT-PCR 10,11克隆。蛇毒蛋白的鉴定也可以通过整合来自毒腺cDNA文库15,21产生的序列数据库标准质谱技术来实现在高通量方式进行。这样的工作，从毒液和使用上面详述的方案收集腺体开始，显示了它的有效性的一个例子，示于图1。 从L.收集毒液蝽成年女性用胰蛋白酶消化，并进行了在溶液中MuDPIT分析，连接性HPLC（高效液相色谱法），以串联质谱（MS / MS）。这里是由亚利桑那州蛋白质组学联盟大学进行的MuDPIT分析。检测肽的群众和他们的分离片段（子离子，推断来自谱）进行了比较，从从L.构建的基因表达文库中获得的cDNA序列翻译理论预测的肽序列金星毒腺（通过解剖协议被收购的腺体如上所述）21。在该实验中，36蛋白从所有收集的光谱检测，在99.9％的概率阈值，并且包含至少一个检测到的肽。由43总频谱（ 图1A中所示范例谱）所支持的一个检测到的蛋白质的，对应于三个独家肽，覆盖所述蛋白质序列（ 图1B）的35％。所检测的蛋白质（来自collecte翻译<html> ðcDNA中）具有顶部经BLASTp命中从L.到latrodectin在用在该实验中检测到的蛋白质的氨基酸序列的水平斑寇蛛 （GenBank登录P49125.1），与电子分2e的-46，并共享80％的同一性。 Latrodectin，其也被称为阿尔法- latrotoxin低分子量的蛋白质，是黑寡妇的一个公认的毒液组分蜘蛛22，23，验证所提出的协议的有效性。从毒液确定了一些蛋白质对应于对于没​​有爆击被发现的序列。目前还不清楚这样的结果是否是由于可用于蜘蛛有限的基因组资源以及其蛋白的有限功能性的信息，或者因为未知的蛋白质代表毒液污染物。然而，基因或蛋白质表达分析研究这种新的蛋白质的毒腺相对于其它组织应该揭示它是否是一个真正的毒液组分的丰度。 ENT“FO：保together.within页=”总是“&gt; <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51618/51618fig1highres.jpg" width="600" /> 图1：使用质谱法从黑寡妇毒液确定Latrodectin肽（A）代表光谱（43 1）通过L的MuDPIT分析MS / MS检测部分。 蝽其被分配到在B部分光谱中所示的收集cDNA序列预测的翻译毒液表示质荷检测到的子离子的比（m / z）的对由分段母体肽（CGEEDFGEEIVK）产生的水平轴，以在顶端的信表明基于子离子质量的肽序列。（B）蛋白质序列的顺序进行光谱A部分被分配，显示为红色，加粗和下划线的文字从A部分所示的光谱，额外的红色文字（不加粗）对应的序列代表检测与其它收集SP在这种蛋白质另一种肽ectra。蛋白质序列是从解剖毒腺获得的cDNA序列翻译。

Watch this Scientific Journal Video about Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses at JoVE.com

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

本文提供了一个协议，使用电刺激蜘蛛毒液为了提取1）进行蛋白质鉴定，2）刺激毒腺基因的表达; 3）执行毒液的功能研究。这之后的毒腺microdissections用于基因表达研究的描述。

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

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33.1K 次观看.  University of Massachusetts Lowell. 本文提供了一个协议，使用电刺激蜘蛛毒液为了提取1）进行蛋白质鉴定，2）刺激毒腺基因的表达; 3）执行毒液的功能研究。这之后的毒腺microdissections用于基因表达研究的描述。 

视频: Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

科研

生物学

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

从人窦卵泡中提取、标记和纯化谱系特异性细胞

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

饲养巴西黄蝎子提取毒液

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

作者聚焦：优化蝎子毒液提取以生产抗蛇毒血清 

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

用于抗蛇毒血清生产的 Tityus serrulatus （巴西黄蝎子）的圈养维持和毒液提取

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...