Name: analysis of epididymal protein synthesis and secretion
Uploaded: 2018-08-25T15:00:00.000+00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about Analysis of Epididymal Protein Synthesis and Secretion at JoVE.com

提交人要感谢澳大利亚国家卫生和医学研究理事会项目赠款 APP1103176 支持这项工作。

所有涉及动物组织收集的实验程序都是由纽卡斯尔大学动物保育和伦理委员会批准的。
1. 石蜡嵌入附睾切片的免疫荧光染色 (图1和 2)
<ol><li>在成年小鼠安乐死后, 通过 CO2吸入 (瑞士老鼠, 超过8周), 仔细解剖附睾 (使用手术剪刀和镊子) 没有覆盖结缔组织和脂肪, 浸泡在 Bouin 的固定器解决方案 (> 十倍体积/组织重量) 过夜固定。</li>
	<li>每天用70% 乙醇与2×改变2天, 然后通过分级乙醇 (70%、95% 和 100%) 脱水, 准备入渗和嵌入石蜡块。</li>
	<li>切片的石蜡块在厚度为4-6 µm 和安装在幻灯片上, 准备免疫荧光染色。</li>
	<li>在油烟机中, 脱蜡的附睾石蜡切片添加足够的二甲苯到滑瓶完全浸入组织部分 (3×5分钟每次)。</li>
	<li>水化的组织切片浸泡在分级乙醇溶液稀释纯化 H2O (100% 乙醇5分钟, 100% 乙醇5分钟, 90% 乙醇1分钟, 80% 乙醇1分钟, 70% 乙醇1分钟, 50% 乙醇1分钟)。</li>
	<li>用足够的磷酸盐缓冲盐水 (PBS) 将一个滑动罐中的切片洗涤5分钟, 以完全浸入整个组织部分 (按照这些指示进行后续洗涤)。</li>
	<li>醒酒适当的抗原检索解决方案 (i., 10 毫摩尔柠檬酸钠, 50 毫摩尔/升三 pH 10.5 或替代抗原检索解决方案, 取决于要检测的抗原) 成一个滑动架和微波直到沸腾。将幻灯片浸入此解决方案中, 并将组织切片与热诱导抗原检索条件优化为单个抗体 (见表 1)。 注意: 在抗原检索过程中, 确保幻灯片完全沉浸在抗原检索溶液中。</li>
	<li>从微波炉中取出滑动容器, 冷却至室温。</li>
	<li>用 PBS 冲洗幻灯片, 用一支液态的滑动标记笔在组织切片周围进行跟踪。</li>
	<li>将幻灯片放在一个加湿的容器中 (由容器底部的湿润组织创建), 并应用阻塞解决方案 (以前通过0.45 µm 过滤器过滤的 3% BSA/PBS), 1 小时37摄氏度。</li>
	<li>用 PBS 冲洗一次幻灯片。</li>
	<li>用适当的原抗体稀释到实验性的 1% BSA/PBS 在4°c 过夜 (1:60 用于 anti-DNM1、DNM2 和 DNM3 抗体; 1:100 anti-ATP6V1B1 抗体, 见材料表抗体详细信息)。 注意: 要区分特定于非特异抗体的结合, 必须包括严格的阴性 (i.、二次抗体、原发性抗体 preabsorbed 免疫肽) 和阳性对照24。</li>
	<li>复温在室温下放置30分钟的幻灯片。</li>
	<li>在震动平台 (60 rpm) 上用 PBS 清洗幻灯片3×每10分钟。</li>
	<li>用适当的二级抗体稀释在 1% BSA/PBS (过滤通过0.45 µm 过滤器) 在37°c 为 1 h (1:400 稀释为所有次要抗体, 看见抗体细节的材料表)。 注意: 从这个步骤开始, 将幻灯片容器保持在黑暗中。对于双标记, 选择一个兼容的二次抗体组合 (i. e., 二级抗体必须在不同的物种中提出)。</li>
	<li>在震动平台 (60 rpm) 上用 PBS 清洗幻灯片3×每10分钟。</li>
	<li>Counterstain 碘碘化物 (PI, 7.48 μmol/升) 或 4΄, 6-diamidino-2-苯基吲哚 (DAPI, 4.37 μmol/升) 在室温下的2分钟, 以标记细胞核。</li>
	<li>每两次在震动平台 (60 rpm) 上用 PBS 清洗幻灯片5分钟。</li>
	<li>装上 10% Mowiol 4-88 的部分, 在30% 甘油的溶液中制备0.2 摩尔/升三 (pH 8.5) 和 2.5% 1, 4-杂双环-(2.2. 2)-辛烷值。</li>
	<li>用指甲油封住盖玻片, 将幻灯片存放在4摄氏度以供将来观察。 注意: 建议在准备后尽快执行幻灯片的成像, 以避免过多的荧光损失。</li>
</ol>2从小鼠附睾中分离 Epididymosomes (图 3)
<ol><li>在成年老鼠安乐死以后通过 CO2吸入 (瑞士老鼠8星期老), 灌注他们的血管与 PBS (预热到37°c) 减少附睾组织的血液污染。 注意: 血浆含有不同的外来体种群, 其大小与 epididymosomes25相似。从附睾组织的血液清除效果可以通过检查最初的片段, 一个高度血管化的附睾段位于接近额段 (i. e.,图 1A中的1区)。</li>
	<li>仔细解剖没有上覆脂肪和结缔组织的附睾, 用改良的比格斯、惠顿和 Whittingham 培养基冲洗 (BWW; pH 值 7.4, 渗透压300毫摩尔/千克水26,27), 以减少任何表面电位血液污染。</li>
	<li>涂抹附睾组织去除多余的培养基, 解剖该附睾 (e.,图 1A中的2-5 区), 并转移到含有 BWW 培养基的新鲜培养皿 (35 x 10 毫米)。确保介质数量足以进行最终恢复。 注: 对于6位附睾, 建议使用1.1 毫升的培养基, 以允许恢复900µL, 然后均匀分裂, 并应用在2预准备的梯度之上 (见步骤 2.9)。</li>
	<li>用剃刀刀片将一些小切口插入到头部的组织中。不要切碎组织, 从而避免污染样品与过多胞浆含量。在37°c 的30分钟内, 将含有组织的轻度搅动的钢板孵化, 释放腔内的内容。</li>
	<li>过滤结果悬浮通过70µm 膜去除细胞碎片。</li>
	<li>收集滤液, 并将其与连续离心步骤在4°c, 以提高速度, 以消除细胞碎片 (i., 500 x g, 2000 x g, 4000 x g, 8000 x g, 5 分钟每个;1.7万 x g为20分钟, 最后 1.7万 x g为另外10分钟或直到没有颗粒在离心以后形成)。 注意: 在初始 500 x g离心步骤后对颗粒的颜色进行评估是很重要的, 以确保最小的血液污染存在。丢弃此颗粒显示粉红色着色的任何样品。</li>
	<li>通过稀释密度梯度介质 (包括 60% (w/v) 水 iodixanol), 用0.25 摩尔/iodixanol 蔗糖和10毫摩尔/升三 (pH 7.5) 来制备不连续的梯度 (包括40%、20%、10%、5% 层)。</li>
	<li>在 ultracentrifuge 管 (11 x 35 毫米) 上准备渐变, 每个分数为450µL (图 3)。观察每个分数应用后的渐变, 以确保在加载附睾流体样本之前, 每个层之间成功地形成界面。在使用当天准备每一个渐变, 但是, 在加载前, 附睾腔内流体样品可保存在4摄氏度至2小时。</li>
	<li>小心地添加450µL 的附睾腔内液体悬浮 (对应于从3附睾的头部收集的材料) 在单一梯度之上。</li>
	<li>Ultracentrifuge 梯度在 16万 x g在4°c 为 18 h。 注意: 由于这种离心是在非常高的速度进行, 所有 ultracentrifuge 管必须配对和平衡精确。检查管, 以确保他们没有任何明显的损害, 可能危及他们的完整性。</li>
	<li>轻轻地移除12个相等的分数 (每个由185µL 组成) 从最上层开始, 朝着梯度的底部前进。如果适用 (最多两个渐变), 则池中从每个渐变中恢复的等效分数。 注: 鼠标 epididymosomes 是最丰富的分数 9-1122, 见图 4和讨论。</li>
	<li>在回收和汇集 9-11 分, 稀释成2毫升的 PBS, 并 ultracentrifuge 样品在 10万 x g在4摄氏度 3 h (13 x 56 毫米管) 颗粒的 epididymosomes。 注意: 由于 epididymosome 颗粒很难看到, 确保管的方向被注意到, 因为它们被放置在转子和标记的管, 以表明预期的位置, epididymosome 颗粒。确保每管有足够的容积 (i., 超过其总容量的 50%), 以防止管道坍塌的风险。</li>
	<li>小心地吸入和丢弃上清, 不干扰 epididymosome 颗粒。</li>
	<li>评估 epididymosome 纯度 (图 4)。</li>
	<li>根据下游应用并用重悬 epididymosome 颗粒进入所需介质。例如, BWW 培养基一般用于与精子共孵化的实验, 或者是一个适当的裂解缓冲剂, 用于为附睾蛋白质组通过 SDS 页的分辨率进行准备。</li>
</ol>3. mECap18 细胞的免疫荧光染色
<ol><li>无菌盖玻片的制备 (在细胞培养罩中进行)<ol>
<li>将盖玻片 (12 x 12 毫米) 浸泡在70% 乙醇中10分钟, 在乙醇灯以上高温下干燥消毒。</li>
		<li>冷却的盖玻片在十年代转到12井板块。</li>
		<li>应用无菌聚 l-赖氨酸溶液覆盖盖玻片, 室温下沉淀10分钟。</li>
		<li>放弃聚 l-赖氨酸溶液, 用不育的 H2O 或适当的培养基冲洗盖玻片。</li>
	</ol>
</li>
	<li>制备 mECap18 细胞<ol>
<li>通过 2 x 105 mECap18 细胞的整除数在12井板的每个井包含盖玻片。</li>
		<li>培养细胞与 mECap18 细胞培养基 (DMEM 补充 1% l-谷氨酰胺, 1% 丙酮酸钠, 1% 青霉素/链霉素, 50 μmol/升 5α-androstan-17β-3-oneC-IIIN), 在10% 摄氏度的恒温箱中含有37胎小牛血清 (血清) 5%共2夜。</li>
		<li>一旦细胞坚持盖玻片, 丢弃培养基, 并用 PBS 冲洗两次细胞。</li>
		<li>添加足够数量的4% 多聚甲醛 (粉煤灰) 稀释在 PBS, 以浸泡整个盖玻片和固定在室温下的细胞15分钟。</li>
		<li>丢弃粉煤灰溶液, 在 PBS 中冲洗两次盖玻片。</li>
	</ol>
</li>
	<li>免疫荧光染色<ol>
<li>Permeabilize mECap18 细胞浸泡在0.1% 海卫 X-100 在 PBS 10 分钟。</li>
		<li>用 PBS 冲洗盖玻片。</li>
		<li>阻断 mECap18 细胞与 3% BSA, 并进行 immunolabeling 的细胞使用等效的协议, 为附睾组织切片描述。</li>
	</ol>
</li>
</ol>4. 从条件细胞培养培养基中分离蛋白质
<ol><li>条件细胞培养培养基的收集<ol>
<li>通过整除数 4 x 105 mECap18 细胞在6井板每井与 mECap18 细胞培养基补充10% 个血清 24 h。</li>
		<li>用 mECap18 细胞培养基 (无血清制备) 清洗 mECap18 细胞三次, 以去除残留的血清和任何相关的蛋白质污染物。</li>
		<li>加入1.5 毫升的 mECap18 细胞培养基 (无血清制备), 并在 5% CO2下的37°c 孵化器中与 mECap18 细胞孵育12小时。 注: mECap18 细胞在这一步可以评估不同的目标抗原根据实验设计。</li>
		<li>经过12小时的孵化, 收集细胞培养基和离心机在 2000 x g 10 分钟, 以消除所有细胞碎片。 注: 孵化期可根据实验设计/终点评估, 并考虑细胞对应用治疗的耐受性而改变。建议根据特定的实验方案调整孵化时间, 以确保达到最佳效果。</li>
		<li>通过应用标准的台盼蓝排斥试验评估 mECap18 细胞的生存能力28。丢弃所有细胞存活能力下降到90% 以下的物质, 以消除由死或垂死细胞释放的蛋白质所引入的偏倚。</li>
		<li>将蛋白质从细胞培养基中分离出来, 或保持培养基在-80 摄氏度。</li>
	</ol>
</li>
	<li>蛋白质隔离 (在油烟机中进行)<ol>
<li>添加20% 体积的冷冻100% 乙酸酸到80% 容量的条件细胞培养基, 沉淀从培养的 mECap18 细胞释放的蛋白质。以恒定的混合, 夜间孵化4摄氏度。</li>
		<li>孵化后, 颗粒沉淀蛋白离心 (17, 000 x克, 4 °c 10 分钟)。 注: 由于所需的蛋白质数量有限, 在培养基中可能会使颗粒在离心后不易形象化。因此, 必须在离心前正确定位管, 并注意不要在清除清液时干扰预期颗粒位置。</li>
		<li>在再离心之前, 先用冷冻丙酮将上清液和两次颗粒洗净 (17, 000 x克, 4 °c 10 分钟)。</li>
		<li>在空气干燥之前, 要小心地除去并丢弃上清液, 然后在通风罩内残留丙酮。</li>
		<li>并用重悬蛋白质颗粒在适当的提取缓冲器准备终点分析, 以检测完整的分泌蛋白剖面和/或单个靶蛋白 (e., SDS 页, 免疫印迹法)。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

这些研究纳入了使用 Bouin 的固定附睾组织, 已受到石蜡嵌入和标准切片协议。Bouin 的固定液包括甲醛、苦味酸酸和乙酸的混合物, 每个组分具有特定的互补作用。因此, 甲醛与原胺反应形成蛋白交联, 苦味酸酸慢慢穿透组织形成的盐, 从而使碱性蛋白凝固, 反之, 醋酸迅速穿透组织, 导致凝血核酸。这些组合的性质产生了 Bouin 作为一个固定的选择保存的形态学细节, 它的使用被广泛报道的附睾文献。?...

所有哺乳动物的精子都有可能显示正向渐进运动, 并通过附睾 (雄性额外睾丸导管系统的高度专门化区域) 受精卵子, 这可能用 7-14 天的时间来导航 (取决于物种)1。由于父亲染色质的极度凝结和大部分细胞质的脱落, 伴随着睾丸内精子的 cytodifferentiation, 它们随后的功能成熟完全由它们的相互作用驱动。附睾微环境。这一环境反过来, 由内衬附睾的分泌和吸收活动创造, 并显示一个特殊水平的段段变化1。因此, 在蛋白质合成和分泌方面, 最活跃的部分是位于附睾近端的部分 (即, 额和语料库)2。这项活动反映精子的功能轮廓, 细胞首先开始显示功能能力的特征 (i. e., 渐进动力和绑定到酸可溶性透明糖蛋白的能力) 后, 其通过3。这些功能属性继续发展之前达到最佳水平, 因为精子到达远端附睾段 (马尾), 其中他们储存在一个静止状态, 准备射精。这种精子储存库的形成和维持也与衬里上皮紧密相连, 在马尾中由强吸收活性4、5控制。虽然解剖差异已报告6,7,8, 这种分区分工似乎是一个特点的附睾, 是共享的大多数哺乳动物物种研究至今, 包括我们自己的9,10。事实上, 从临床的角度来看, 附睾功能障碍对男性因素不孕11的病因有重要的贡献, 因此突出了了解这种特殊组织的调节的重要性。
因此, 令人遗憾的是, 我们对附睾生理的理解, 以及调控精子成熟和贮存的顺序阶段的机制, 仍有待完全解决。在促成因素中, 附睾研究的进展受到限制, 这种组织的总体复杂性和对其管腔微环境的调控机制的认识。解剖上, 我们知道, 除了额位, 语料库和马尾段的区别, 附睾可以进一步细分为几个区域 (图 1A), 每隔12间隔, 并以基因/蛋白的离散剖面为特征表达式13,14,15,16,17,18。事实上, 根据对附睾节段基因表达的详细转录分析, 老鼠和大鼠模型中有多达6和9个不同的附睾区, 分别为19、20。这种复杂性大概反映了附睾躯体的组成, 假上皮包括许多不同的细胞类型;每种不同的, 他们的丰度, 分布和分泌/吸收活动的长度。因此, 主细胞是迄今为止最丰富的附睾细胞类型, 构成了所有上皮细胞的80% 以上。因此, 主要细胞负责附睾蛋白生物合成和分泌物的大部分5。与此相反, 作为附睾躯体内第二大细胞类型的透明细胞种群, 主要参与腔内成分的选择性吸收和这种微环境的酸化5。添加另一层复杂性, 雄激素和其他 lumicrine 因素的睾丸起源施加差异控制每一个这些附睾细胞类型取决于他们的定位沿道。
尽管这种复杂性所造成的限制, 仍有重大进展, 以解决附睾功能的机制基础。这些研究的一个关键是应用先进的质谱策略建立广泛的附睾蛋白质组的清单, 并与从这些初步调查中选择的单个蛋白质进行详细分析。这一方法的一个例证是我们最近在老鼠模型21中对 mechanoenzymes DNM 家族的描述。我们最初对 DNM 的兴趣是由它在 endocytotic 过程耦合中的双重作用所推动的。在这些观察的基础上, 我们能够证明 DNM (DNM1-DNM3) 的三规范亚型在小鼠附睾中有高度表达, 并适当定位以在蛋白质分泌和吸收中发挥调节作用21.此外, 我们能够清楚地区分每个 DNM 异构体的基础上, 他们的细胞和亚细胞定位, 从而表明, 他们拥有互补, 而不是多余的活动, 在附睾上皮21。
在这里, 我们描述了用于研究小鼠附睾中 DNM 表达的实验方法, 希望这些信息能在其他附睾蛋白的鉴定中得到更广泛的应用, 从而有助于我们了解男性生殖道这一重要元素的功能。具体来说, 我们描述了在石蜡嵌入附睾切片中靶蛋白免疫荧光标记的稳健方法的发展, 以及随后通过免疫荧光检测这些蛋白质的空间分布。显微镜。我们进一步记录我们最近优化的协议22 , 以隔离和表征 epididymosomes;小的外切状囊泡, 构成附睾分泌剖面的关键元素, 在促进精子成熟23中似乎起着突出的作用。作为补充的方法, 我们还描述了免疫荧光检测的目标蛋白在永生化鼠额附睾上皮 (mECap18) 细胞系和利用这个资源作为一个模型, 以探讨对附睾的调节体外分泌活性。

<table><tbody><tr><td>Dynamin 1 antibody</td><td>Abcam</td><td>ab108458</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rabbit, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Dynamin 2 antibody</td><td>Santa Cruz</td><td>sc-6400</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Dynamin 3 antibody</td><td>Proteintech</td><td>14737-1-AP</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rabbit, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>ATP6V1B1 antibody</td><td>Santa Cruz</td><td>sc-21206</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>CD9 antibody</td><td>BD Pharmingen</td><td>553758</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: monoclonal</td></tr><tr><td>Flotillin-1 antibody</td><td>Sigma</td><td>F1180</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rabbit, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>ALOX15 antibody</td><td>Abcam</td><td>ab80221</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rabbit, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>TUBB antibody</td><td>Santa Cruz</td><td>sc-5274</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Mouse, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: monoclonal</td></tr><tr><td>PSMD7 antibody</td><td>Abcam</td><td>ab11436</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Rabbit, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Rabbit Alexa Fluor 488</td><td>Thermo</td><td>A11008</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Goat Alexa Fluor 488</td><td>Thermo</td><td>A11055</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Donkey, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Goat Alexa Fluor 594</td><td>Thermo</td><td>A11058</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Donkey, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Rat Alexa Fluor 594</td><td>Thermo</td><td>A11007</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Rabbit HRP</td><td>Millipore</td><td>DC03L</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Rat HRP</td><td>Millipore</td><td>DC01L</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>Anti Mouse HRP</td><td>Santa Cruz</td><td>sc-2005</td><td>&lt;strong&gt;Host species&lt;/strong&gt;: Goat, &lt;strong&gt;Isotype&lt;/strong&gt;: IgG, &lt;strong&gt;Class&lt;/strong&gt;: polyclonal</td></tr><tr><td>4&#039;, 6-diamidino-2-phenylindole (DAPI)</td><td>Sigma</td><td>D9564</td><td></td></tr><tr><td>propidium iodide (PI)</td><td>Sigma</td><td>P4170</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Bovogen</td><td>SFBS-F</td><td></td></tr><tr><td>DMEM</td><td>Thermo</td><td>11960-044</td><td></td></tr><tr><td>L-glutamine</td><td>Thermo</td><td>25030-081</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Thermo</td><td>15140-122</td><td></td></tr><tr><td>5&amp;alpha;-androstan-17&amp;beta;-ol-3-oneC-IIIN</td><td>Sigma</td><td>A8380</td><td></td></tr><tr><td>sodium pyruvate</td><td>Thermo</td><td>11360-070</td><td></td></tr><tr><td>Trypsin-ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Xylene</td><td>VWR Chemicals</td><td>1330-20-7</td><td></td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>64-17-5</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>Tris</td><td>Astral</td><td>0497-5KG</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>1, 4-diazabicyclo-(2.2.2)-octane</td><td>Sigma</td><td>D2522</td><td></td></tr><tr><td>Poly-L-gysine</td><td>Sigma</td><td>P4832</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>78787</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T6146</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma</td><td>T9159</td><td></td></tr><tr><td>Acetone</td><td>Ajax Finechem</td><td>A6-2.5 L GL</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Poly (vinyl alcohol)</td><td>Sigma</td><td>P8136</td><td></td></tr><tr><td>D-Glucose</td><td>Ajax Finechem</td><td>783-500G</td><td></td></tr><tr><td>OptiPrep Density Gradient Medium</td><td>Sigma</td><td>D1556</td><td></td></tr><tr><td>Fluorescence microscopy</td><td>Zeiss</td><td>Zeiss Axio Imager A1</td><td></td></tr><tr><td>Ultracentrifuge</td><td>BECKMAN COULTER</td><td>Optima Max-XP</td><td></td></tr><tr><td>Microcentrifuges</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Incubator</td><td>Heracell</td><td>150</td><td></td></tr><tr><td>Large Orbital Shaker</td><td>Ratek</td><td>OM7</td><td></td></tr><tr><td>Microwave</td><td>LG</td><td>MS3840SR /00</td><td></td></tr><tr><td>Lab pH Meter</td><td>MeterLab</td><td>PHM220</td><td></td></tr><tr><td>Liquid-repellent slide marker</td><td>Daido Sangyo</td><td>Mini</td><td></td></tr><tr><td>Coverslip</td><td>Thermo</td><td>586</td><td></td></tr><tr><td>6 well plate</td><td>CELLSTAR</td><td>657160</td><td></td></tr><tr><td>12 well plate</td><td>CELLSTAR</td><td>665180</td><td></td></tr><tr><td>Slide</td><td>Mikro-Glass</td><td>SF41296PLMK</td><td></td></tr><tr><td>0.45 &amp;micro;m filter</td><td>Millox-HV</td><td>SLHV033RS</td><td></td></tr><tr><td>Kimwipes Dustfree Paper</td><td>KIMTECH</td><td>34155</td><td></td></tr><tr><td>Ultracentrifuge tube (2.2 ml, 11 &amp;times; 35 mm)</td><td>BECKMAN COULTER</td><td>347356</td><td></td></tr><tr><td>Ultracentrifuge tube (3.2 ml, 13 &amp;times; 56 mm)</td><td>BECKMAN COULTER</td><td>362305</td><td></td></tr><tr><td>Cell strainer 70 &amp;micro;m Nylon</td><td>FALCON</td><td>352350</td><td></td></tr><tr><td>Petri dish 35 &amp;times; 10 mm with cams</td><td>SARSTED</td><td>82.1135.500</td><td></td></tr><tr><td>Slide jar</td><td>TRAJAN</td><td>#23 319 00</td><td></td></tr></tbody></table>

analysis of epididymal protein synthesis and secretion

哺乳动物附睾生成任何内分泌腺体中最复杂的腔内液体之一, 以支持睾丸后成熟和精子的贮存。这种复杂性的产生是由于衬里上皮细胞的分泌和吸收活性的综合作用。在这里, 我们描述的技术, 分析附睾蛋白合成和分泌的重点是模型蛋白家族的动力蛋白 (DNM) mechanoenzymes;具有调节双向膜贩运活动潜力的大型 GTPases。为了研究附睾组织中蛋白质的表达, 我们描述了石蜡嵌入切片中靶蛋白免疫荧光标记的鲁棒方法, 并通过对这些蛋白质的空间分布进行了检测。免疫荧光显微镜。我们还描述了最优化的方法, 以分离和鉴定的外切像囊泡, 称为 epididymosomes, 这是分泌到附睾腔参与细胞间沟通与成熟的精子细胞。作为补充方法, 我们还描述了免疫荧光检测的目标蛋白在 SV40-immortalized 小鼠附睾上皮 (mECap18) 细胞系。此外, 我们还讨论了 mECap18 细胞系作为一种合适的体外模型, 用于研究附睾分泌活动的调节。为此, 我们描述了维持 mECap18 细胞系的培养要求和使用有选择性的药理抑制方案, 它们能够影响其分泌蛋白的分布。后者很容易通过收获的条件培养基, 分泌蛋白的浓度通过乙酸酸/丙酮沉淀和他们随后的分析通过 SDS 页和免疫印迹法。我们认为, 这些联合方法适合于分析替代附睾蛋白靶点, 作为确定它们在精子成熟和/或贮存中功能作用的前奏。

图 1和图 2显示了小鼠附睾 DNM 免疫荧光定位的代表性结果。调查的三 DNM 亚型中的每个都显示不同的定位配置文件。因此, DNM1 的特点是相对温和的弥漫性标记的附睾细胞在整个初始段和额附睾 (图 2A)。相比之下, DNM2 异构体首先检测到在最初的部分细胞的对立的基底和顶端边界附近, 然后被重?...

Watch this Scientific Journal Video about Analysis of Epididymal Protein Synthesis and Secretion at JoVE.com

Analysis of Epididymal Protein Synthesis and Secretion

在这里, 我们报告动力蛋白的免疫荧光定位, 以说明在石蜡嵌入小鼠附睾切片和永生化附睾细胞系 (mECap18) 的蛋白质检测协议。我们还描述了从附睾液和条件细胞培养基中分离分泌蛋白的协议。

附睾蛋白的合成与分泌分析

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

analysis-of-epididymal-protein-synthesis-and-secretion

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  University of Newcastle. 在这里, 我们报告动力蛋白的免疫荧光定位, 以说明在石蜡嵌入小鼠附睾切片和永生化附睾细胞系 (mECap18) 的蛋白质检测协议。我们还描述了从附睾液和条件细胞培养基中分离分泌蛋白的协议。

视频: Analysis of Epididymal Protein Synthesis and Secretion

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

科研

生物化学

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

发育生物学

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

医学

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...