Name: phloem sap sampling from brassica napus for 3d-page of protein and ribonucleoprotein complexes
Uploaded: 2018-01-09T15:00:00.000+00:00
Duration: 11 min 23 s
Description: Watch this Scientific Journal Video about Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes at JoVE.com

我们感谢詹妮弗大叔和 Urszula Zarzenska 的科学支持。这项工作是由欧洲委员会在第七框架计划, 赠款 LFF-GK06 ' DELIGRAH ' (Landesforschungsförderung 汉堡), 和 DFG 补助金 (DFG 柯 856_6-1) 的职业融合补助金 (烟, PCIG14-GA-2013-63 0734) 的财政支持。授予 JK。

1. 从乙型油菜植物中提取韧皮部汁液的取样和制备4,8,20
<ol><li>对于韧皮部 sap 取样, 使用充足8周老的B 型油菜植物, 只显示最初开花, 以防止花粉污染。</li>
	<li>使用皮下注射针 (Ø0.8 毫米) 和点状花序茎多次。重复在其他几个花序茎和其他植物获得足够的韧皮部 sap (图 2a)。 注: 对于复杂的分析, 建议从至少600µL 韧皮部 sap 开始。4到5株植物在韧皮部汁液的200和700µL 之间将产生。</li>
	<li>用滤纸擦拭受伤部位的第一滴。这些水滴主要含有来自周围受损组织的高污染物质, 需要丢弃。</li>
	<li>收集以下滴通过移和存储收集的渗出物在一个 pre-chilled 圆锥1.5 毫升反应管在-20 ° c 使用 ice-free 冷却系统。</li>
	<li>继续收集, 直到没有进一步的下落形成是可观察的, 但不长于 1 h。这一过程可以重复两天后, 没有大量的收集韧皮部 sap 损失。 注: 所收集的韧皮部汁液可立即使用或储存在-80 ° c, 以备将来分析。在-80 ° c 贮存时, 将反应管在液氮中冻结。</li>
	<li>将韧皮部样品浓缩为大约1/6 的初始容积的th , 使用离心聚光器, 分子量 (分子) 为1万道尔顿 (Da)。离心浓缩样品在4° c 和 2万 x g 为至少15分钟去除尘土微粒。 注意: 韧皮部汁液浓度不会导致大量蛋白质的流失。</li>
	<li>对于后续的复杂分析, 请继续执行步骤3.1。</li>
</ol>2. 韧皮部 sap 纯度控制使用逆转录 pcr (rt-pcr) 4,8,20
<ol><li>用标准的 rna 萃取方法分离韧皮部汁液中的总 rna (例如TRIzol 或苯酚-氯仿萃取, 然后从水相的异丙醇沉淀和乙醇洗涤步骤根据制造商的协议)。 注意: 苯酚-氯仿是有毒的。工作在一个敞篷与适当的个人防护设备之下。提取的 RNA 在-80 ° c 可以被存放在酒精在六月。</li>
	<li>通过消化与 dnasei I (1 u/µg) 在37° c 中去除 DNA 的污染, 并将 EDTA 添加到最终浓度为5毫米, 并在75° c 处孵育样品, 10 分钟, 使酶钝化。</li>
	<li>要获得纯 rna, 净化和浓缩样品的另一个纯化步骤 (例如使用 RNA 清理套件, 根据制造商的说明)。</li>
	<li>采用逆转录 pcr (rt-pcr) 技术合成 cdna 合成试剂盒, 使用 150 ng 纯 RNA, 如制造商协议所述。 注: 该 cDNA 可贮存在-20 ° c, 直至进一步使用。</li>
	<li>使用5µL 的 cDNA 作为标准 PCR 反应的模板, 由制造商指定, 以放大特定的记录和检查琼脂糖凝胶电泳。用 rubisco 小亚基、蛋白 h 和花粉皮蛋白 (图 1b,表 1) 的引物来确认韧皮部样品的纯度。</li>
</ol>3. 蓝色本机页4,25,26,31
<ol><li>使用 self-poured 本机梯度凝胶, 如在其他地方所描述的27或商业双三梯度凝胶。</li>
	<li>负载20到30µg 的浓缩蛋白样品每井到凝胶至少在 triplicates。</li>
	<li>使用深蓝色阴极缓冲 B 和阳极缓冲器 (表 2) 在4° c 和 150 V 上运行凝胶, 直到样品通过了凝胶的 1/3rd (ca 20 到30分钟,图 3a)。 注: 对于北印迹, 一个单一的凝胶车道是足够的, 而为分析的蛋白质组成, 由3D 页和质谱, 建议将同一波段从多达十凝胶车道, 以集中较少丰富的蛋白质。</li>
	<li>暂停运行并交换深蓝色阴极缓冲 B 与浅蓝色阴极缓冲 B/10 (表 2) 并继续运行。完成凝胶运行时, 蓝色的前面开始洗的凝胶 (ca 120 到 180 min,图 3a)。 注: 成品蓝原胶可在4° c 贮存至少一周。长时间储存会导致扩散带, 应避免。深蓝色缓冲器 B 可重复使用。</li>
</ol>4. 可选: 使用蓝色原生北印迹检测 RNA 4
<ol><li>切出不同的乐队或使用整个凝胶车道从蓝色本地凝胶和转移到尼龙膜的半干印迹在3.5 毫安/cm2为60分钟, 并标记上和下凝胶边界与一支铅笔在膜上 (图 4a)。</li>
	<li>印迹后, 用 DEPC 处理过的水冲洗膜, 在两个滤纸之间晾干。</li>
	<li>用12万µJ/cm2 (八十五年代如果在材料表中使用交) 对涂抹膜进行 UV 交联。</li>
	<li>预杂交膜与6毫升杂交缓冲 (商业或自制) 60 分钟在68° c 在杂交烤箱 (图 4a)。</li>
	<li>添加一个额外的1毫升杂交缓冲包含4µL 3 的化探针 (100 nM)。</li>
	<li>用探针在一夜之间孵化膜, 同时将烤箱从68° c 冷却到37° c。</li>
	<li>用含有 2x SSC 和 0.1% SDS 的缓冲液冲洗膜, 在室温下5分钟去除探针空泛。 谨慎: SDS 会引起过敏。在使用 sds 和 sds 解决方案时, 要戴上手套和工作服。</li>
	<li>使用亲和-hrp 共轭和鲁米诺作为辣根过氧化物酶 (hrp) 基质, 对膜上的3化探针进行检测, 从而产生一个敏感的化学发光反应 ( 图 4a)。</li>
</ol>5. 第二维: 三聚丙烯-尿素页4,28
<ol><li>从蓝色原生凝胶中切除单个波段。结合在平行车道上运行的样本中的波段来实现复杂蛋白质的进一步浓缩 (图 5)。 注: 切除带可储存在单一反应管, 直到进一步使用在4° c。</li>
	<li>倒入12% 的三聚丙烯脲凝胶 (厚度1毫米, 6.8cm x 8.6 厘米 (宽 x 长))。准备10毫升的凝胶溶液 A 的分离凝胶 (表 2), 倒在两个玻璃板和覆盖与异丙醇。在聚合完成后除去异丙醇, 并将4毫升的凝胶溶液 B (表 2) 转移到凝胶上, 并插入10井梳。 警告: Unpolymerized 丙烯酰胺可以是神经毒性和 TEMED 是有害的, 如果吸入。始终佩戴个人防护设备, 并在遮光罩下工作。</li>
	<li>将切除的凝胶带转移到1.5 毫升的反应管中, 并为凝胶件 (图 5,表 2) 的平衡度添加1毫升的 2x SDS 采样缓冲器。<ol>
<li>在室温下孵育样品10分钟, 先在加热块 (95 ° c) 或微波中煮沸, 再在室温下再孵育样品15分钟。</li>
		<li>堆叠几个平衡凝胶件代表同一复杂到一个单一的凝胶口袋。</li>
		<li>执行电泳使用阳极和阴极运行缓冲器在 150 V 45 至60分钟和消费整个凝胶车道。</li>
	</ol>
</li>
	<li>在酸性缓冲液中孵育20分钟 (100 毫米三, 100 毫米醋酸) 和平衡在125毫米的三盐酸, pH 6.8 为另 20 min (图 5)。</li>
</ol>6. 第三个维度: SDS-页4,32
<ol><li>根据 Laemmli32 (厚度: 1.5 毫米, 6.8cm x 8.6 厘米 (宽度 x 长度)) (表 2) 将 15% SDS 分离凝胶, 并在上面添加一层4% 堆积凝胶。 警告: SDS、丙烯酰胺和 TEMED 是有毒和有害的。工作在一个敞篷和佩带个人保护设备。</li>
	<li>将平衡凝胶车道转移到 sds 凝胶上, 用 0.5% (w/w) 琼脂糖在 1x sds 运行缓冲液中覆盖凝胶, 并辅以溴蓝的微量 (图 5)。</li>
	<li>用琼脂糖在微波前加载到凝胶上煮沸 SDS 运行缓冲器。</li>
	<li>为了运行一个蛋白质标记来估计单组分的分子重量, 在凝胶的一端插入一张滤纸, 直到琼脂糖凝固或使用一种特殊的梳子, 通常用于 IPG 凝胶。</li>
	<li>将凝胶在150伏处运行60分钟, 用胶体马斯亮、银渍或任何其它适合随后质谱分析的染色方法染色。</li>
	<li>利用基质辅助激光解吸/电离飞行时间 (MALDI) 或 LC 质谱法等质谱方法分析可见的蛋白质斑点。 注: 我们建议使用胶体马斯亮染色, 如已描述的33。在我们手中, 即使是较不丰富的蛋白质, 也可以充分染色, 并允许质谱分析与合理的数据质量。</li>
</ol>

<ol>
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作者没有什么可透露的。

韧皮部是一个高利益的车厢, 因为它构成了同化的主要运输路线, 并且是重要的在不同的植物零件之间的长途信号9,20,34, 35。除了小分子, 大分子, 如蛋白质和 rna 已经牵连到韧皮部维护和信号4,7,8。对韧皮部组成的分析受限于在大多数植物物种的韧皮部样品的有限的可及性。昆虫针技术是广泛适用的, 但实验要求和结果在极少量的韧皮部样本11。一个简单的技术用于韧皮部取样是 EDTA 促进渗出12。EDTA 螯合物价离子如钙, 从而防止了 P 蛋白和胼胝关闭筛板。它易于使用, 但容易受到损坏的细胞和样品被稀释的污染。通过 EDTA 消耗价离子也可能导致现有络合物的分解。但是, 它允许从种类繁多的物种中取样, 包括模型植物a. 南芥15。韧皮部汁液的自发渗出只发生在少数植物种类中。它是一种侵入性的方法, 涉及严重的植物组织损伤10。我们最近建立了B. 型油菜作为研究长途运输的模型物种4820。在这里, 韧皮部 sap 可以从小切口取样, 从而减少伤人的影响和污染源。
采用不同取样技术, 对不同植物韧皮部样品的蛋白质组进行了调查, 最近几年7,8,17,3637, 38,39。对于这些蛋白质组研究, 蛋白质被提取和沉淀在变性条件下, 因此不允许关于蛋白质的结论: 蛋白质和蛋白质: 在本机条件下存在的核酸相互作用。沉淀 (CoIP) 和叠加化验表明, 一个主要的核糖复合物可能存在于韧皮部 sap 从南瓜40, 但它没有证明, 任何大分子复合体在本机条件下存在韧皮部系统。
蓝色本机页, 由 Schägger et al.介绍26, 结合随后的高分辨率凝胶电泳步骤, 可以分离大型蛋白复合物的单个成分。使用3页对本机B. 型油菜韧皮部渗出物的分析, 我们最近可以证明, 在韧皮部样本中存在着几种高分子的蛋白质和 RNP 络合物, 它们是酶活动的4。分离蛋白质复合物的其他方法和 RNPs 象大小排除色谱可能存在, 但在决议方面的失败与 BN 页。此外, 对于一个合理的复杂分离质量, 尺寸排除柱矩阵必须根据所研究的整体复杂分子量进行调整。因此, 分析不同尺寸的复合体将需要不同类型的柱, 这是成本密集型的, 不允许高聚焦力作为 BN 页面。
从B. 型油菜中分析配合物的关键步骤包括用作起始原料的韧皮部汁液总量。至少600µL 的韧皮部样品是必要的, 可以从五充足植物中获得。对于3页和 bn 北印迹有必要启动初始 bn 凝胶与足够数量的韧皮部样本。为了达到这个目的, 韧皮部样品被集中到20到30µg 的蛋白质可以被装入每个井, 并且至少 triplicates 同样样品是并行运行的。过低或过高的浓度会降低对复合物的检测 (图 3)。韧皮部样品需要收集到冰上的反应管, 并应储存冷冻以备以后使用, 以避免蛋白质的降解和周转。蛋白酶抑制剂已发现在所有韧皮部蛋白质分析, 但也完全活跃蛋白酶体描述的韧皮部的B. 型油菜4。此外, 韧皮部样品的体积和组成取决于取样时点和环境条件10。因此, 必须在同样的条件下, 在一天的同一时间收集。压力处理 (例如干旱) 可以减少可以收集的韧皮部的数量。通过质谱法鉴别单蛋白, 必须随时佩戴手套来限制可能的角蛋白污染。在质量指标分析中, 角蛋白导致额外的信号, 并阻碍蛋白质的鉴定。在较高的电压或环境温度下运行 BN 页会导致凝胶的加热, 从而可能导致易碎络合物的拆卸。
这里提出的协议适用于蛋白质的分析: 蛋白质复合物。我们还表明, 它可以用来检测在平行的配合物中包含的特定 rna。为了实现这一点, 我们最近推出了一个协议为 BN 北印迹4。rna 容易被核糖核酸污染物降解。要限制这种降解 DEPC 处理, 应使用水。
所提出的方法的主要局限性包括估计的蛋白质复合物的分子量, 由 BN 页分离, 因为迁移行为取决于大小, 形状, 甚至对配合物的后修改31, 41。由于核酸对迁移行为的影响, RNP 络合物的尺寸估计更为复杂。它也不能阻止相同大小的复合 co-migrate 在一个单一的波段。这可能导致对复杂成分的错误预测。因此, 验证与互补技术的观察到的交互,例如通过功能检测4可能是必要的。并且蛋白质仅微弱地或瞬时地伴生到一个大分子复合体可能丢失在使用的 BN 缓冲。为了避免这种情况, 样品可以交联, 什么也可以导致文物或可以防止蛋白质的鉴定, 或缓冲条件可以优化。
与古典 2 d 页对比, 尿素和 SDS 页的组合允许分离根据疏水性和分子重量而不是等电点 (pI) 和分子量28, 并且不限制分离对蛋白质特定的 pI 范围。相似于古典 2 d 页, 分离的蛋白质是可看见的作为唯一斑点, 但在对角线4,28 (图 5, 图 6)。更多疏水性蛋白质出现在斑点在这对角线之上, 而更多亲水性蛋白质在线之下被发现28。该协议中使用的聚丙烯酰胺浓度将蛋白质分离25至 80 kDa 井。为了使较小或更大的蛋白质分离, 聚丙烯酰胺的浓度可以变化或梯度凝胶可用于第三维度。用3维页 (图 6) 分隔的复杂 IV (图 3) 的组成部分被切除, 胰蛋白酶被消化, 并通过质谱分析。这导致了几个 tRNA 酶的识别。其他复合体的组件最近已发布4。我们的3页启用了不同酶的分离, 即天门冬氨酸、胺、苏氨酸、赖氨酸和甘氨酸酶, 具有相似的分子量 (表 3)。使用一个蛋氨酸 tRNA 特定的探针, 我们能够检测到在复杂 IV 中可能绑定的 tRNA, 但如果全长 tRNA 或 tRNA 片段是在复杂的, 不能与使用的探头区分。要检查核酸是否真的在复杂的范围内, 而不是 co-migrating, 蛋白酶消化韧皮部 sap 样本可能作为适当的迁移控制。
早先推测蛋白质翻译可能发生在韧皮部筛子管之内, 因为几乎多数组分整个核糖体并且伸长因素在韧皮部样品被发现了4,7。我们对 tRNA 和 tRNA 酶的发现似乎支持这个想法。然而, 来自南瓜的韧皮部样品中的 tRNA 片段已被证明是有效的翻译抑制剂42和功能核糖体似乎没有4, 表明翻译不能发生。
综合起来, 这里提出的协议允许分离原生蛋白质: 蛋白质, 甚至 RNP 配合物从韧皮部样品和分离和鉴定各自的组分以高决议。该方法的应用使得在韧皮部样品中发现了新的蛋白质和 RNP 配合物, 因此可以在这一高度专门化的植物室中阐明新型功能。

韧皮部的长距离导管对于高等植物中有机养分的分配是必不可少的, 但也参与调节许多对生长发育、病原体防御和适应不良的不同过程。环境条件。除了像离子、激素或代谢物本身这样的小的效应外, 像蛋白质和 rna 的大分子最近也成为了潜在的信号。
研究表明, 韧皮部负荷的蛋白质是大小依赖和控制的大小排除限制 (SEL) 的孔-plasmodesmal 单位连接的同伴细胞 (CC) 和筛元素 (SE), 通常在范围10至 > 67 kDa1,2,3. 然而, 尽管有这些大小的限制, 在韧皮部系统中发现了较大的蛋白质和蛋白质复合物, 挑战大小排除的想法4, 甚至从 CC 到 SE 的蛋白质的非特异性损失被建议5.
复合以及大型核糖复合体在生物合成和维持细胞结构完整性方面起着关键作用6。有证据表明, 韧皮部移动蛋白蛋白和核糖配合物存在4,7, 但对其功能的了解很少。在韧皮部筛子元素蛋白质: 蛋白质和 RNP 复合体被牵连了与长途运输并且/或者信号8,9。为了解开移位配合物的功能, 需要在不同的环境或应力条件下研究它们的组成。为了实现这一目标, 必须将原生复合体隔离, 并以足够的分辨率分离它们的成分。
为了从韧皮部中分离出高分子重量的配合物, 首先需要获得足够的质量和数量的 sap 样品。可用于韧皮部取样的技术取决于感兴趣的植物种类。三主要方法获取韧皮部汁液存在10: 昆虫 stylectomy11, EDTA 促进渗出12, 和自发渗出13。
来自拟南芥 (A. 芥) 的韧皮部样品, 全球实验室的主要模型工厂, 只能通过 EDTA 辅助渗出或蚜虫 stylectomy1415获得。这两种方法都导致了高度稀释的韧皮部汁液或非常低的样本量。EDTA 促进渗出也容易受到污染, 可能不代表真正的韧皮部组成16。自发韧皮部渗出仅限于植物物种, 如瓜, 羽扇或丝兰, 在那里切断植物部分导致的韧皮部 sap 自发排放, 可以很容易地收集13,17,18, 19。我们最近建立了油菜 (甘蓝型油菜) 作为韧皮部分析的一个合适的模型系统, 因为它是一个近亲的a. 南芥和几个升的韧皮部 sap 可以获得从小切口, 已表明高纯度4,8,20。此外, 在 2014年21中发布了B. 型油菜基因组序列。
除了蚜虫 stylectomy, 所有的韧皮部收集方法都包括在取样现场对周围组织的破坏, 韧皮部汁液的组成可能会因伤害而改变。因此, 无论采用何种取样方法, 都必须检查韧皮部样品的质量。有不同的方法为收集的韧皮部汁液的质量控制,例如通过核糖核酸活动的决心22,23, RT PCR 与引物反对 Rubisco8,24, 或分析糖组成8。
不同的分离和分离蛋白质复合物的方法可以应用, 包括大小排斥色谱, 蔗糖密度离心, 或样品过滤通过膜与不同的分子量削减取舍。但是, 这些方法不允许高分辨率。该技术称为蓝色本机页 (BN 页), 首先由 Schägger et al描述。25在分辨率方面优于。它被成功地用来确定从各种细胞器或蜂窝裂解及其相对丰度26,27的大型原生蛋白复合物的分子量。
为了进一步阐明蛋白质复合物的复杂成分, 有必要在变性条件下分离各组分, 导致复杂组分的完全拆卸。这可以通过 sds 页的第二个维度28,29或, 以实现更高的分辨率, 通过第二维的等电聚焦 (隐含), 其次是 sds 页30的第三维度, 并随后识别使用质谱法的蛋白质。
在本协议中, 我们描述了从乙型油菜植物中提取的纯韧皮部液的取样, 并利用3D 电泳法结合 BN-聚丙烯-尿素-页和 SDS 页分析了这些韧皮部样品中的蛋白质复合物。分离的蛋白质复合组分随后被辨认用质谱法。此外, 我们引入蓝色原生北印迹作为一种方法, 允许检测在大型 RNP 配合物4中的 rna, 扩大了该方法的适用性。

<table><tbody><tr><td>1.5 mL reaction tubes</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>10 well comb for SDS PAGE, thickness 1 mm</td><td>BioRad</td><td>1653359</td><td></td></tr><tr><td>2-Propanol</td><td>AppliChem</td><td>131090</td><td></td></tr><tr><td>30 % Acrylamide-bisacrylamide solution (29:1) solution</td><td>BioRad</td><td>1610156</td><td></td></tr><tr><td>30 % Acrylamide-bisacrylamide solution (37.5:1) solution</td><td>BioRad</td><td>1610158</td><td></td></tr><tr><td>96 % Ethanol</td><td>Carl Roth</td><td>P075</td><td></td></tr><tr><td>Acetic acid</td><td>Carl Roth</td><td>6755</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Aluminum sulfate-(14-18)-hydrate</td><td>AppliChem</td><td>141101</td><td></td></tr><tr><td>Ammonium peroxodisulfate (APS)</td><td>Carl Roth</td><td>9592.3</td><td></td></tr><tr><td>Bis-Tris</td><td>AppliChem</td><td>A3992</td><td></td></tr><tr><td>Boric acid</td><td>AppliChem</td><td>A2940</td><td></td></tr><tr><td>Bromophenol blue</td><td>AppliChem</td><td>A2331</td><td></td></tr><tr><td>Centrifugal concentrators &lt;em&gt;e.g.&lt;/em&gt; Vivaspin 500 (Mwco 10 kDa)</td><td>Sartorius</td><td>VS0102</td><td></td></tr><tr><td>Chemiluminescent Nucleic Acid Detection Module Kit</td><td>Thermo Fisher Scientific</td><td>89880</td><td></td></tr><tr><td>Chromatography papers &lt;em&gt;e.g.&lt;/em&gt; 3 mm CHR</td><td>Whatman</td><td>3030-153</td><td></td></tr><tr><td>CoolBox M30 System</td><td>Biocision</td><td>BCS-133</td><td></td></tr><tr><td>Coomassie brilliant Blue G-250</td><td>AppliChem</td><td>A3480</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>AppliChem</td><td>A0881</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AppliChem</td><td>A1101</td><td></td></tr><tr><td>DNase I</td><td>AppliChem</td><td>A3778</td><td></td></tr><tr><td>Ethanol abs.</td><td>AppliChem</td><td>A3693</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>AppliChem</td><td>A1103</td><td></td></tr><tr><td>Gel Imaging system &lt;em&gt;e.g.&lt;/em&gt; Chemidoc Touch</td><td>BioRad</td><td>1708370</td><td></td></tr><tr><td>GeneRuler 1kb plus</td><td>Thermo Fisher Scientific</td><td>SM1331</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem</td><td>141339</td><td></td></tr><tr><td>Glycine</td><td>AppliChem</td><td>131340</td><td></td></tr><tr><td>Heating block TS1</td><td>Biometra</td><td>846-051-500</td><td></td></tr><tr><td>Hybridization oven &lt;em&gt;e.g.&lt;/em&gt; GFL Hybridization Incubator 7601</td><td>GFL</td><td>7601</td><td></td></tr><tr><td>Hypodermic needle (0.8 mm)</td><td>B Braun</td><td>4658309</td><td></td></tr><tr><td>IPG gel comb, thickness 1 mm</td><td>BioRad</td><td>1653367</td><td></td></tr><tr><td>Labeling of RNA &lt;em&gt;e.g.&lt;/em&gt;Biotin 3&#039;end DNA Labeling Kit</td><td>Thermo Fisher Scientific</td><td>89818</td><td></td></tr><tr><td>Native gradient gel &lt;em&gt;e.g.&lt;/em&gt; Novex NativePAGE 4&amp;ndash;16% Bis-Tris protein gel</td><td>Invitrogen</td><td>BN1002BOX</td><td></td></tr><tr><td>Nylon membrane &lt;em&gt;e.g.&lt;/em&gt; Hybond-N+</td><td>Amersham Pharmacia</td><td>RPN203B</td><td></td></tr><tr><td>Ortho-phosphoric acid</td><td>Carl Roth</td><td>6366.1</td><td></td></tr><tr><td>PageRuler prestained protein ladder</td><td>Thermo Fisher Scientific</td><td>26616</td><td></td></tr><tr><td>Power supply for Gel electrophoresis EPS</td><td>Amersham Pharmacia</td><td>18-1130-01</td><td>available from GE Healthcare</td></tr><tr><td>RNA Cleanup Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Semi dry blot &lt;em&gt;e.g&lt;/em&gt;. Fastblot 43B semi dry Blot</td><td>Biometra</td><td>846-015-100</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>AppliChem</td><td>A1149</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>AppliChem</td><td>142363</td><td></td></tr><tr><td>Synthesis of cDNA &lt;em&gt;e.g.&lt;/em&gt; RevertAid First Strand cDNA Synthesis Kit</td><td>Thermo Fisher Scientific</td><td>K1621</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>AppliChem</td><td>A1148</td><td></td></tr><tr><td>Tricine</td><td>AppliChem</td><td>A3954</td><td></td></tr><tr><td>Tris</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>Tri-sodium citrate dihydrate</td><td>AppliChem</td><td>A3901</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596026</td><td></td></tr><tr><td>ULTRAhyb Ultrasensitive Hybridization Buffer</td><td>Thermo Fisher Scientific</td><td>AM8670</td><td></td></tr><tr><td>Urea</td><td>AppliChem</td><td>A1360</td><td></td></tr><tr><td>UV Crosslinker &lt;em&gt;e.g.&lt;/em&gt; Stratalinker 2400</td><td>Agilent</td><td>NC0477671</td><td>from Fisher Scientific</td></tr><tr><td>Vertical electrophoresis apparatus &lt;em&gt;e.g.&lt;/em&gt;The XCell SureLock Mini-Cell or Mini-Protean III System</td><td>Thermo Fisher Scientific or BioRad</td><td>EI0001 or 1658004</td><td></td></tr><tr><td>Wipers &lt;em&gt;e.g.&lt;/em&gt; KIMWIPES Delicate Task Wipers</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr></tbody></table>

phloem sap sampling from brassica napus for 3d-page of protein and ribonucleoprotein complexes

对高等植物的韧皮部进行取样往往是很费力的, 而且对植物种类的依赖性很大。然而, 在变性条件下的蛋白质组研究可以在不同的植物物种中实现。原生蛋白: 蛋白质和蛋白质: 韧皮部样品中的核酸配合物尚未被分析, 尽管它们可能在维持这一专门的隔间或长距离信号中发挥重要作用。大型分子组装可以使用蓝色的本地凝胶电泳 (BN 页) 来隔离。它们的蛋白质组分可以用随后的十二烷基硫酸钠页 (SDS 页) 分开。然而, 蛋白质具有相似的分子量 co-migrate, 什么可以阻碍蛋白质的鉴定通过质谱。结合 BN 页与两种不同的变性凝胶电泳步骤, 即三聚丙烯尿素和 SDS 页, 使蛋白质的额外分离, 根据其亲水性/疏水性, 从而提高分辨率和成功的蛋白质鉴定。它甚至允许区分的蛋白质, 只有不同的后的修改。此外, 蓝色本地印迹可用于识别大分子复合物中的 RNA 成分。我们表明, 我们的协议是适当的, 以解开蛋白质和 RNA 成分的本地蛋白质: 蛋白质和核糖 (RNP) 化合物发生在韧皮部样本。将蓝色的本机页面与两个不同的变性页面相结合, 可以帮助分离不同种类的大型蛋白质复合物, 并通过质谱法提高其组分的识别率。此外, 该协议还具有足够的鲁棒性, 可以同时检测单个蛋白复合物中的潜在束缚核酸。

在这里, 我们提出了一个协议, 允许蛋白质分析: 蛋白质和蛋白质: RNA 配合物的韧皮部样本从甘蓝型油菜。工作流在图 1中进行了说明。B. 型油菜在其他模型生物体上的一个主要优势是相对容易的韧皮部样品收集从小穿孔在花序茎。这允许获得相当大数量的纯韧皮部样品。在取样韧皮部时, 最好检查污染物, 例如通过 rt-pcr8。在图 2中描述了从纯韧皮部样本中获得的代表性结果。不受污染的韧皮部样品应显示蛋白 h 的可见带, 而没有 rubisco 和花粉皮蛋白的信号出现。rubisco 小亚基可以作为一个控制的样本从树叶, 花序茎或花粉。蛋白 h 在所有样品中都可以检测到, 因为它的 mRNA 在不同种类的韧皮部中发现, 而花粉皮蛋白转录物只应在花芽标本中可见。当韧皮部取样是在完全开花的植物 (花粉皮蛋白) 中或当韧皮部取样穿刺的第一个液滴未被正确丢弃 (rubisco) 时, 就会产生污染物。
在 BN 页之前它是强制的集中韧皮部汁液。使用20至30µg 的蛋白质每井, 至少四主要蛋白复合带变得可见后 BN 页的B. 油菜韧皮部 sap, 过低或太高的浓度不允许明确分离的配合物 (图 3)。建议在二维凝胶的一个单一的口袋中装载几个代表同一复合体的凝胶带, 以进一步集中每个单一复合体的蛋白质, 用于下游加工和随后的质谱鉴别。
为了识别韧皮部大分子络合物的单个成分, 我们将初始 BN 页与第二维聚丙烯-尿素和第三维 SDS-页结合起来, 以实现单个蛋白质的分离。在尿素和 SDS-page 凝胶中, 由于蛋白质迁移行为的不同, 可以观察到高分辨率的分离。通常, 属于一个单一的复合体的蛋白质将是可见的, 作为对角线横跨凝胶。这条线上的蛋白质有增加疏水性, 而在线之下的蛋白质代表更多亲水部分28 (图 5,图 6)。
对于 RNP 配合物的表征, 我们使用了 bn 页凝胶的一部分来执行 bn 北印迹。在将整个车道印迹到尼龙膜上并通过 UV 辐射进行交联后, 可以使用 rna 特定的探针对 rna 进行可视化, 如图 4所示。在这里, 它表明, 一个特定的 tRNA 只存在于一个特定的复杂。这种 tRNA 结合复合物的蛋白质组分可以用上述3D 凝胶方法进一步研究。质谱分析表明, 该复合物主要包含 tRNA-酶, 证实了 BN 北印迹的 tRNA 结合活性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57097/57097fig1.jpg"> 图 1: 油籽油菜韧皮部汁液中核糖配合物分析的工作流程在对韧皮部样品进行取样和制备后, 采用 BN-页来分离原生复合物。这种 bn-页凝胶允许对核酸进行平行分析, 并通过质谱法对络合物的蛋白质组分进行鉴定。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57097/57097fig2.jpg"> 图 2:甘蓝型油菜韧皮部汁液的取样和纯度控制在用无菌皮下针穿刺 8-10 周老油菜植株的花序梗后, 用吸管收集渗出液并将其转移到 ice-cold 反应管 (a), 以确定韧皮部样品的纯度,在组织特异性样品中放大蛋白 h、rubisco 小亚基和花粉皮蛋白的转录 (b) 无反转录酶的 rt-pcr 作为对照 (-) 进行。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57097/57097fig3.jpg"> 图 3: BN 页。B. 型油菜的浓缩韧皮部样品的蓝原生页的示意图表示。(a) 加载梯度蛋白凝胶与15-20 µL 的浓缩韧皮部 sap, BN 页是执行与1x 深蓝色阴极缓冲 B 在 150 V 20-30 分钟在一个寒冷的房间。然后缓冲被交换反对1x 淡蓝色负极缓冲 B/10, 并且页完成在 150 V 为 120-180 分钟直到深蓝奔跑前面用尽胶凝体。BN-页凝胶显示四不同的带代表四个高分子重量复合体。(b) 加载过少 (c) 或太多 (d) 韧皮部蛋白可降低单个复合物的可见度。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57097/57097fig4.jpg"> 图 4: BN 北印迹.BN 北印迹方法的示意图表示。(a) BN 页的车道通过半干印迹转移到尼龙膜上。经过紫外交联和、, 膜是孵化与 RNA 特异性探针 (这里的蛋氨酸 tRNA 特异性探头), 这是化在 3 '-年底在杂交烤箱过夜。自由探针通过洗涤步骤被去除, 并且 RNA 的检测由亲和-HRP 共轭和鲁米诺进行。使用这种方法, 我们观察到, 复杂 IV 从 BN 页包含蛋氨酸 tRNA。(b) 马斯亮染色凝胶和 tRNA 印迹类似两个不同的凝胶车道平行运行, 以避免迁移的差异。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57097/57097fig5.jpg"> 图 5: 3 页.3 d 页方法的示意图表示法。从同一个复合体的几个波段从 BN 页凝胶中切除, 在载入缓冲液中孵育, 并转移到第二维聚丙烯-尿素-页的一个井中。电泳后, 将整个凝胶道切掉, 在酸性酸缓冲液中洗净, 平衡, 放在 15% SDS 页凝胶上。在第三维度页之后, 用质谱法对分离的蛋白质进行马斯亮染色和分析。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57097/57097fig6.jpg"> 图 6: 3 页后的代表性结果.在第一个维度之内 BN-页几复合体出现。他们的蛋白质成分可以进一步分离两个后续的变性页, 导致一个对角斑点模式。<a href="//cloudflare.jove.com/files/ftp_upload/57097/57097fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>探讨</td>
			<td>序列</td>
		</tr>
<tr>
<td>蛋白 h</td>
			<td>
 转发: CTCAAGGCAGCCAAAGAATC 反转: ATGGCCTGAACATCGAACTC</td>
		</tr>
<tr>
<td>Rubisco 小链</td>
			<td>
 转发: TTCACCGGCTTGAAGTCATC 反转: CCGGGTACTCCTTCTTGCAT</td>
		</tr>
<tr>
<td>花粉皮蛋白 BRU77666</td>
			<td>
 转发: TCAGAACTGGAGCTTCAACG 反转: TTCCTTAATGGCCTCAGTGG</td>
		</tr>
</tbody></table>表 1: 韧皮部样品纯度控制用底漆.为确定韧皮部样品纯度 rubisco 小链、蛋白 h 和花粉皮蛋白的引物, 可用于 cDNA 扩增。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>缓冲区和解决方案</td>
			<td colspan="2">内容</td>
			<td>评论</td>
		</tr>
<tr>
<td>深蓝色阴极缓冲器 B</td>
			<td colspan="2">15毫米双三酸7。0 50毫米聚丙烯 0.02% (w/v) 马斯亮蓝色 G250</td>
			<td>配方由 Fiala et al. 17改编 pH 值应调整到7。0 缓冲器可以做为10x 库存</td>
		</tr>
<tr>
<td>浅蓝色阴极缓冲 B/10</td>
			<td colspan="2">15毫米双三酸7。0 50毫米聚丙烯 0.002% (w/v) 马斯亮蓝色 G250</td>
			<td>用阴极缓冲液稀释阴极缓冲 B, 无需马斯亮, 即可制备缓冲器。</td>
		</tr>
<tr>
<td>阳极缓冲器</td>
			<td colspan="2">
 50毫米双三酸7。0</td>
			<td>缓冲器可作为10x 库存解决方案进行准备</td>
		</tr>
<tr>
<td>2x SDS 样本缓冲器</td>
			<td colspan="2">
 125毫米三盐酸盐 pH 值6。8 4% SDS 20% 甘油 0.2 M 数码的 0.02% (w/v) 溴蓝色</td>
			<td></td>
		</tr>
<tr>
<td>传输缓冲区1x 缓冲区</td>
			<td colspan="2">
 89毫米三 pH 7。6 89 mM 硼酸 2毫米 EDTA</td>
			<td>缓冲器可作为5x 或10x 的库存进行存储。使用无核糖核酸的去离子水是很重要的。</td>
		</tr>
<tr>
<td>2x SSC 缓冲</td>
			<td colspan="2">
 300毫米氯化钠 30毫米 Na3柠檬酸盐 pH 值7。0</td>
			<td></td>
		</tr>
<tr>
<td>3x 凝胶缓冲液</td>
			<td colspan="2">
 3 M 三 1 M HCl 0.3% SDS pH 值8.45</td>
			<td>从 Schägger 19改编的食谱
</td>
		</tr>
<tr>
<td>三聚丙烯凝胶溶液</td>
			<td colspan="2">
 10毫升: 30% 丙烯酰胺-bisacrylamide (29:1) 3.34 mL 6 M 尿素 3x 凝胶缓冲3.34 毫升 70% 甘油1.4 毫升 将 ddH2O 添加到 10 mL 10% APS 40 µL TEMED 4 µL</td>
			<td>重6米尿素, 加入3x 凝胶缓冲液, 丙烯酰胺-bisacrylamide 溶液和 70% gycerol。在水浴加热到60° c 溶解尿素。添加 ddH2O 到10毫升, 10% APS, 最后 TEMED 聚合。</td>
		</tr>
<tr>
<td>三聚丙烯凝胶溶液 B</td>
			<td colspan="2">
 6毫升: 30% 丙烯酰胺-bisacrylamide (29:1) 0.8 mL 6 M 尿素 3x 凝胶缓冲1.5 毫升 将 ddH2O 添加到 6 mL 10% APS 45 µL TEMED 4.5 µL</td>
			<td>重6米尿素, 加入3x 凝胶缓冲液和丙烯酰胺-bisacrylamide 溶液。在水浴加热到60° c 溶解尿素。添加 ddH2O 到10毫升, 10% APS, 最后 TEMED 聚合。</td>
		</tr>
<tr>
<td>1x 三聚丙烯运行缓冲器 (阳极)</td>
			<td colspan="2">
 100毫米三 22.5 毫米 HCl pH 值8。9</td>
			<td>从 Schägger 19改编的食谱
</td>
		</tr>
<tr>
<td>1x 三聚丙烯运行缓冲器 (阴极)</td>
			<td colspan="2">
 100毫米三 100毫米聚丙烯 1% SDS pH 值8.25</td>
			<td>从 Schägger 19改编的食谱
</td>
		</tr>
<tr>
<td>酸性缓冲液</td>
			<td colspan="2">
 100毫米三 100毫米乙酸</td>
			<td></td>
		</tr>
<tr>
<td>4% SDS 堆积凝胶</td>
			<td colspan="2">
 2毫升: ddH2O 1.4 mL 30% 丙烯酰胺-bisacrylamide (37.5: 1) 0.33 毫升 1 M 三 pH 6.8 0.25 毫升 10% SDS 20 µL 10% APS 20 µL TEMED 2 µL</td>
			<td></td>
		</tr>
<tr>
<td>15% SDS 分离凝胶</td>
			<td colspan="2">
 10毫升: ddH2O 2.3 mL 30% 丙烯酰胺-bisacrylamide (37.5: 1) 5 毫升 1. 5 米, pH 8.8 2.5 毫升 10% SDS 100 µL 10% APS 100 µL TEMED 4 µL</td>
			<td></td>
		</tr>
<tr>
<td>1x SDS 运行缓冲器</td>
			<td colspan="2">
 25毫米三 192毫米甘氨酸 0.1% SDS</td>
			<td></td>
		</tr>
<tr>
<td>马斯亮染色液</td>
			<td colspan="2">
 5% (w/v) 硫酸铝-(14-18)-水合物 10% (v/v) 乙醇 (96%) 2% (v/v) 正磷酸酸 (85%) 0.02% (w/v) 马斯亮亮蓝色 G-250 
			 </td>
			<td></td>
		</tr>
<tr>
<td>ULTRAhyb 灵敏杂交缓冲</td>
			<td colspan="2"></td>
			<td>Ambion, 生命技术</td>
		</tr>
</tbody></table>表 2: 解决方案和缓冲配方.3 d 页分析所需的所有缓冲区和解决方案的列表。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>现货号</td>
			<td>兆瓦 obs [kDa]</td>
			<td colspan="2">识别</td>
			<td>有机体</td>
			<td>加入号</td>
			<td>MW [kDa]</td>
			<td>吉祥物评分</td>
		</tr>
<tr>
<td>CIV_1</td>
			<td>130</td>
			<td colspan="2">假定抗病蛋白 At4g19050</td>
			<td>B. 油菜</td>
			<td>CDX78917</td>
			<td>131。1</td>
			<td>110</td>
		</tr>
<tr>
<td>CIV_2</td>
			<td>130</td>
			<td colspan="2">假定抗病蛋白 At4g19050</td>
			<td>B. 油菜</td>
			<td>CDX78917</td>
			<td>131。1</td>
			<td>89</td>
		</tr>
<tr>
<td>CIV_3</td>
			<td>100</td>
			<td colspan="2">热休克 70 kDa 蛋白14样</td>
			<td>B. 油菜</td>
			<td>XP_013748865</td>
			<td>89。9</td>
			<td>113</td>
		</tr>
<tr>
<td>CIV_4</td>
			<td>90</td>
			<td colspan="2">真核伸长因子2 细胞分裂控制蛋白48同源 A</td>
			<td>B. 油菜 B. 油菜</td>
			<td>CDX90241 CDY41316。1</td>
			<td>89。5 89。5</td>
			<td>111 197</td>
		</tr>
<tr>
<td>CIV_5</td>
			<td>85</td>
			<td colspan="2">热休克蛋白90-2 样</td>
			<td>B. 复活节岛</td>
			<td>XP_009132342</td>
			<td>79。8</td>
			<td>172</td>
		</tr>
<tr>
<td>CIV_6</td>
			<td>80</td>
			<td colspan="2">苏氨酸--tRNA 连接 甘氨酸--tRNA 连接</td>
			<td>B。 B. 油菜</td>
			<td>XP_013599115 CDY43195</td>
			<td>81。5 80。8</td>
			<td>110 88</td>
		</tr>
<tr>
<td>CIV_7</td>
			<td>70</td>
			<td colspan="2">热休克蛋白 70 kDa 赖氨酸--tRNA 连接样</td>
			<td>B。 B. 油菜</td>
			<td>XP_013685267 XP_013747530</td>
			<td>67。8 69。9</td>
			<td>230 150</td>
		</tr>
<tr>
<td>CIV_8</td>
			<td>65</td>
			<td colspan="2">芥子 天门冬氨酸--tRNA 连接2 胺--tRNA 连接1</td>
			<td>B. 油菜 B. 油菜 B. 油菜</td>
			<td>ABQ42337 XP_013670803 XP_013729126</td>
			<td>60。6 61。6 63。5</td>
			<td>183 141 153</td>
		</tr>
<tr>
<td>CIV_9</td>
			<td>60</td>
			<td colspan="2">芥子</td>
			<td>B. 油菜</td>
			<td>ABQ42337</td>
			<td>60。6</td>
			<td>164</td>
		</tr>
<tr>
<td>CIV_10</td>
			<td>55</td>
			<td colspan="2">Adenosylhomocysteinase 2 样</td>
			<td>B. 复活节岛</td>
			<td>XP_009135865</td>
			<td>53。1</td>
			<td>139</td>
		</tr>
<tr>
<td>CIV_11</td>
			<td>55</td>
			<td colspan="2">伸长系数 1-α1样</td>
			<td>B。</td>
			<td>XP_013586115</td>
			<td>51。9</td>
			<td>161</td>
		</tr>
<tr>
<td>CIV_12</td>
			<td>50</td>
			<td colspan="2">胱氨酸裂解酶 CORI3-like</td>
			<td>B. 油菜</td>
			<td>XP_013653143</td>
			<td>48。1</td>
			<td>237</td>
		</tr>
<tr>
<td>CIV_13</td>
			<td>44</td>
			<td colspan="2">胱氨酸裂解酶 CORI3-like</td>
			<td>B. 复活节岛</td>
			<td>XP_009108611</td>
			<td>48。3</td>
			<td>213</td>
		</tr>
<tr>
<td>CIV_14</td>
			<td>38</td>
			<td colspan="2">果糖--1,6-醛</td>
			<td>B. 复活节岛</td>
			<td>XP_009115993</td>
			<td>38。4</td>
			<td>226</td>
		</tr>
<tr>
<td>CIV_15</td>
			<td>45</td>
			<td colspan="2">胱氨酸裂解酶 CORI3-like</td>
			<td>B. 复活节岛</td>
			<td>XP_009108611</td>
			<td>48。3</td>
			<td>219</td>
		</tr>
<tr>
<td>CIV_16</td>
			<td>45</td>
			<td colspan="2">胱氨酸裂解酶 CORI3</td>
			<td>B. 复活节岛</td>
			<td>CDY69765</td>
			<td>46。9</td>
			<td>209</td>
		</tr>
<tr>
<td>CIV_17</td>
			<td>38</td>
			<td colspan="2">果糖--1,6-醛</td>
			<td>B. 复活节岛</td>
			<td>XP_009115993</td>
			<td>38。4</td>
			<td>124</td>
		</tr>
<tr>
<td>CIV_18</td>
			<td>18</td>
			<td colspan="2">肽-脯顺反式异构酶 CYP18-3-like</td>
			<td>B. 油菜</td>
			<td>XP_009101932</td>
			<td>18。3</td>
			<td>128</td>
		</tr>
<tr>
<td>CIV_19</td>
			<td>45</td>
			<td colspan="2">胱氨酸裂解酶 CORI3-like</td>
			<td>B. 复活节岛</td>
			<td>XP_009108611</td>
			<td>48。3</td>
			<td>177</td>
		</tr>
</tbody></table>表 3: 从复杂 IV. 中识别出的蛋白质组分在 3 d 页后, 在 tRNA 结合体中发现了几种 tRNA 酶和进一步的蛋白质。

Watch this Scientific Journal Video about Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes at JoVE.com

甘蓝型油菜韧皮部汁液取样用于蛋白质和核糖核蛋白复合物的 3D-PAGE |生物学 |视频

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

从甘蓝型油菜中提取3页蛋白质和核糖配合物的韧皮部汁液

在这里, 我们提出了一个协议, 以分析蛋白质组成的大本地蛋白质: 蛋白质和蛋白质: 核酸复合物油菜 (B. 油菜)韧皮部渗出液使用3D 聚丙烯酰胺凝胶电泳 (PAGE) 方法结合蓝色本机 (BN) 与两个变性页后, 质谱鉴定。

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...

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Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Fundamentals

Macromolecules

SCIENTISTS IN ACTION

13.7K 次观看.  Universität Hamburg. 在这里, 我们提出了一个协议, 以分析蛋白质组成的大本地蛋白质: 蛋白质和蛋白质: 核酸复合物油菜 (B. 油菜)韧皮部渗出液使用3D 聚丙烯酰胺凝胶电泳 (PAGE) 方法结合蓝色本机 (BN) 与两个变性页后, 质谱鉴定。

视频: Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

科研

生物化学

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

萝卜品种多糖的 PNGase 研究

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

生物学

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

收集和分析<em&gt;拟南芥</em&gt;韧皮部分泌物使用EDTA促进方法

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

从甘蓝型油菜中提取3页蛋白质和核糖配合物的韧皮部汁液

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