Name: assay for phosphorylation and microtubule binding along with localization of tau protein in colorectal cancer cells
Uploaded: 2017-10-10T12:30:00.000+00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells at JoVE.com

这项研究是作为项目的一部分, 题为 ' 开发和产业化的高价值的海洋微藻的化妆品原料 ', 由海洋和渔业部, 韩国, 并得到了内部资助 (2Z04930) 的支持来自 KIST 江陵天然产物研究所。

1. 试剂的制备 <ol> <li> 添加10和 #181; (1 毫米) 磺氟化物溶液, 10 和 #181; l (1x 从100x 库存) 蛋白酶抑制剂鸡尾酒溶液, 10 和 #181; l (1x 从100x 存货) 磷酸酶抑制剂鸡尾酒解决方案, 以1毫升的 1x radioimmunoprecipitation 检测 (帕) 缓冲区准备完整的帕裂解缓冲液. </li>
	<li> 准备10x 通用蛋白缓冲区 (PEM) 缓冲区. 
	<ol> <li> 添加800毫米 (6.0474 克) 哌嗪-n-氮和 #39;-bis-2-ethanesulfonic 酸, 10 毫米 (95.1 毫克) 乙二醇二 (和 #946;-氨基乙基醚)-n, n, #39;, n-和 #39;-乙酸酸, 10 毫米 (51 毫克) 氯化镁 2 , 1.25 m (3 毫升10米) 氢氧化钠在50毫升管, 用15毫升蒸馏水混合, 用涡流溶解。检查 pH 值 (应为 6.9)。如果 pH 值和 #62; 6.9、丢弃缓冲区并重新改造一个新的批次. 
		注: 氢氧化钠应低于1.25 米, 以确保 pH 值不超过6.9。不建议使用 HCl 来调节 pH 值和 #62; 6.9. </li>
		<li> 添加氢氧化钠滴直到 pH 值为6.9。添加蒸馏水, 使多达25毫升的 10X PEM 缓冲。最后, 使用注射器和 0.2 #181; m 注射器过滤器。将此缓冲器分成等分 1.5 mL 管, 并存储在4和 #176; C. </li>
	</ol> </li> <li> 使用还原剂和染料准备5x 示例缓冲区. 
	<ol> <li> 混合300和 #181; L (60 毫米) 1 米的三盐酸 (pH 6.8), 2.5 毫升 (25%) 50% 的甘油, 1 毫升 (2%) 10% 钠十二烷基硫酸钠 (sds), 和1.2 毫升蒸馏水组成5毫升的 5x SDS 样品缓冲。存储在 #8722; 20 和 #176; C. 
		注: 由于甘油是粘性的, 测量1.25 毫升甘油是困难的。一毫升100% 甘油重1.26 克。因此, 首先在2.5 毫升管中重1.575 克100% 甘油 (相当于1.25 毫升的100% 或50% 毫升的15甘油);与其他组件混合良好. </li>
	</ol> </li> </ol> 2。细胞培养, 用姜黄素或氯化治疗, 并检查蛋白质表达 <ol> <li> 添加〜 1 x 10 6 小贩的116细胞到100毫米组织培养皿中, 含有最低基本培养基 (记忆) 补充10% 胎牛血清 (FBS) 和1% 青霉素-链霉素。经过一天的培养, 细胞将达到60-70% 汇合. 
	注: 所需的车牌数量取决于下面所述的处理数量. </li>
	<li> 当细胞到达 ~ 65% 汇合 (近似地使用显微镜), 去除记忆补充培养基并且增加无血清记忆与5和 #181; m, 10 和 #181; m 和30和 #181; m 姜黄素或25毫米, 50 毫米, 和100毫米氯化。在37和 #176 中孵育, 在含有 5% CO 2 的湿润气氛中为 24 h. </li>
	<li> 24 小时治疗后, 用1毫升 ice-cold 磷酸缓冲盐水 (PBS) 冲洗细胞, 用刮板机刮掉细胞。将刮成的细胞转移到1.5 毫升离心管和离心机4分钟在 1800 x g 在4和 #176; C 获得细胞小球. </li>
	<li> 通过在100和 #181 中悬浮小球来溶解细胞; 在4和 #176 上完成帕缓冲; C 为20分钟, 同时定期敲击管子。简要几种样品. </li>
	<li> 离心机匀在台式离心机在 22570 x g 为20分钟在4和 #176; c. 用吸管将清转移到其他带标签的管子上. </li>
	<li> 测量清中的蛋白质浓度由布拉德福德化验 45 使用商用蛋白质检测试剂盒. </li>
	<li> 用 4X SDS 凝胶缓冲液制备等蛋白质浓度 (10 和 #181; g protein/13 和 #181; L 样本) 的样品。准备好 4x SDS 凝胶-装载缓冲液, 内含400毫米糖 (德勤). 
	注: 在这个阶段, 样品可以存储在和 #8722; 20 和 #176; 如果需要的话. </li>
	<li> 在100和 #176 的热块上孵育样品; C 为 5 min. 用涡流混合样品, 等待冷却10-15 分钟. </li>
	<li> 装配一个电泳系统 。
	<ol> <li> 在 10% sds-聚丙烯酰胺凝胶电泳 (sds 页) 上加载10和 #181; g 蛋白 (13 和 #181; L 样本)。将分子量阶梯放到凝胶上一条车道上. 
		注意: 当电泳开始时, 蛋白质阶梯将分解成具有明显分子重量的蛋白质带。使用这些波段来估计未知蛋白质带的分子量. </li>
		<li> 在加载样品后, 将电泳系统的正负电极连接到电源以保持恒定电压。最初, 从 70 V 开始在20分钟移动蛋白质样品从堆积胶凝体入解决胶凝体。然后, 增加电压到 125 V 大约70-120 分钟, 直到样品和蛋白质梯子到达胶凝体的末端. </li>
		<li> 电泳完成后, 用湿 electroblotting 系统将凝胶转移到聚偏氟 (PVDF) 膜上。转移为〜100分钟在 100 v 块和 #160; 4% 牛血清白蛋白 (BSA) 的膜在室温下为90分钟的封闭缓冲, 大约25和 #176; C. </li>
	</ol> </li> <li> 通过在分子量阶梯的侧面切割它来标记印迹。使用透明的塑料薄片和锋利的切割刀, 以减少印迹。准备主要抗体溶液 (anti-tau 在 1:5, 000; anti-phospho 在 1:4, 000), 并在这个溶液中孵育在4和 #176; C 过夜. </li>
	<li> 在夜间孵育后, 在磷酸盐缓冲 saline-Tween-20 (PBST) 溶液中洗涤 7 min 四倍. </li>
	<li> 准备相关的二级抗体溶液 (山羊兔或 anti-mouse IgG 在 1:5, 000), 并孵化该溶液中的印迹为90分钟, 室温下约25和 #176; c. PBST 四次, 每7分钟洗一次. </li>
	<li> 根据制造商和 #39 的建议, 使用化学发光试剂盒开发印迹。用透明的塑料包装覆盖印迹。通过化学发光成像系统和 #160 获取图像; 用相关软件进行化学发光. </li>
</ol> 3。磷酸酶检测 <ol> <li> 在碱性磷酸酶缓冲中处理细胞裂解 (从步骤 2.5) 到碱性磷酸酶。
	<ol> <li> 对于控件和处理过的示例, 准备20和 #181; 包含20和 #181 的样本; g 总蛋白。添加含有20和 #181 的细胞裂解的体积; g 总蛋白, 其次为2和 #181; 碱性磷酸酶缓冲剂和10和 #181; 碱性磷酸酶。添加蒸馏水, 以弥补20和 #181; L. </li>
	</ol> </li> <li> 在37和 #176 上孵化样品; C 为 1 h. 停止反应, 加入乙酸酸 (EDTA) 在最后浓度为50毫米, 或添加钠钒 (Na 3 VO 4 ) 在最后浓度 10 mm。该反应也可以停止加热75和 #176; C 为 5 min. </li>
	<li> 在样品与磷酸酶的培养完成之前, 准备相同浓度的样品, 而不添加磷酸酶来与磷酸酶处理的样品进行比较. </li>
	<li> 添加新准备的 4X SDS 凝胶缓冲液, 其中包含400毫米的双制式. </li>
	<li> 在100和 #176 的热块上孵育样品; C 为5分钟, 用涡流将样品混合。室温下冷却10-15 分钟. </li>
	<li> 通过 SDS 页组装一个用于运行10% 聚丙烯酰胺凝胶的电泳系统. 
	<ol> <li> 在 10% polyacry 上加载10和 #181; g 蛋白 (13 和 #181; L 样本)。lamide 凝胶。加载分子量阶梯. </li>
		<li> 在加载样本后, 将电泳系统的正负电极连接到电源以保持恒定电压。最初, 从 70 V 开始在20分钟移动蛋白质样品从堆积胶凝体入解决胶凝体。然后, 增加电压到 125 V 和运行的凝胶约70-120 分钟, 直到样品和蛋白质阶梯到达凝胶的末端. </li>
		<li> 电泳完成后, 使用湿 electroblotting 系统将凝胶转移到 PVDF 膜上。转让为〜100分钟在 100 v. </li>
		<li> 通过在分子量阶梯的一侧做一个小切口来标记印迹。使用透明的塑料板和锋利的切割器, 以减少印迹. </li>
	</ol> </li> <li> 块和 #160; 膜和 #160; 4% BSA 在室温下阻塞90分钟的缓冲区. </li>
	<li> 在第一抗体溶液中孵育印迹 (anti-tau 在 1:5, 000) 在4和 #176; C。洗 PBST 四次, 每7分钟的印迹. </li>
	<li> 准备相关的二级抗体溶液 (山羊 anti-mouse IgG 在 1:5, 000), 并孵化在室温下90分钟的印迹。洗涤在 PBST 四次, 每7分钟. </li>
	<li> 根据制造商和 #39 的建议, 使用化学发光试剂盒开发印迹。在透明的塑料包装内覆盖印迹。通过化学发光成像系统获取图像与相关软件. </li>
</ol> 4。微管结合试验 <ol> <li> 对于微管结合试验, 重复步骤 2.1-2.6. </li>
	<li> 准备 1X (2 毫升) pem 缓冲器, 从 10X pem 缓冲区存储在4和 #176; C 通过添加20和 #181; M (40 和 #181; l) 紫杉醇和1毫米 (20 和 #181; l) GTP。从和 #8722 中取出微管 (MT) 的股票; 80 和 #176; c 冷冻机和 大肠杆菌 头工作库存, 从和 #8722; 20 和 #176; c 冷冻机. </li>
	<li> 准备示例, 按 表 1 . </li>
	<li> 孵育在37和 #176; c 为30分钟, Ultracentrifuge 在25和 #176; c 为60分钟, 在 10万 x g . </li>
	<li> 传输120和 #181; 包含未绑定头的清, 用于清洁和标记管. </li>
	<li> 添加120和 #181; L (与上清相同体积) 的5X 样品缓冲液到含有粘合头的颗粒上并彻底混合。将溶液转移到清洁的标签管, 并彻底混合. </li>
	<li> 测量蛋白质浓度 (请参见步骤 2.6). 
	注: 在准备样品时, 使用溶液体积 (步骤 4.6) 来制备颗粒和上清样品. </li>
	<li> 制备等效蛋白质浓度样品, 并加入新制备的 4x SDS 凝胶缓冲液 (含 400 mM). 
	注: 样品可存储在 #8722; 20 和 #176; C 在这个阶段. </li>
	<li> 重复步骤 3.5-3.10. </li>
需要 </ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 样本 </td> <td colspan="2"> 名称 </td> <td colspan="2"> </td> <td colspan="2"> 主要蛋白质需要 </td> <td colspan="2"> PEM-GTP-PTX </td> </tr> <tr> 1 <td> </td> 116 控制 (6.21 和 #956; g/和 #956; l) <td colspan="2"> </td> 2 和 #956; l <td colspan="2">
			<td colspan="2"> 9.66 和 #956; l (60 #956; g) </td>
			<td colspan="2"> 108.34 和 #956; l </td> </tr>
		<tr> <td> 2 </td> <td colspan="2"> 小贩 116-姜黄素 10 & #956; M (4.81 和 #956; g/和 #956; l) </td> <td colspan="2"> 2 和 #956; l </td> <td colspan="2"> 16.63 和 #956; l (80 和 #956; g) </td> <td colspan="2"> 101.37 和 #956; l </td> </tr> <tr> 3 <td> </td>116-姜黄素20和 #956; M (3.28 和 #956; g/和 #956; l) </td> <td colspan="2"> 2 和 #956; l </td> <td colspan="2"> 30.49 和 #956; l (100 和 #956; g) </td> <td colspan="2"> 87.51 和 #956; l </td> </tr> <tr> <td> 4 </td> 氯化 116--25 mM (5.43 和 #956; g/和 #956; l)</td> <td colspan="2"> 2 和 #956; l </td> <td colspan="2"> 18.42 和 #956; l (100 和 #956; g) </td> <td colspan="2"> 99.58 和 #956; </td> </tr> <tr> <td> 5 </td> <td colspan="2"> 大肠杆菌 头 </td> 2 和 #956; l <td colspan="2"> </td> 1 和 #956;tau-352 </td> <td colspan="2"> 117 和 #956; l </td> </tr> <tr> <td> 6 </td> <td colspan="2"> MT </td> <td colspan="2"> 2 和 #956; l </td> <td colspan="2"> X </td> <td colspan="2"> 118 和 #956; l </td> </tr> <tr> <td> </td> <td colspan="2"> </td>2 ">> </td> <td colspan="2"> </td> <td colspan="2"> 120 和 #956; l 每个 </td> </tr> </tbody> </table> 表 1: 微管结合试验的样品制备. 
 5. 姜黄素治疗细胞后的定位和表达 <ol> <li> 使用70% 乙醇清洗片, 使用实验室组织将其烘干。在一个标记为6的组织培养板的每个井中插入一个单一的片。将板放在生物安全柜上, 并在紫外线灯上切换至少3小时. </li>
	<li> 亚文化细胞和种子他们到6个井板的相关井在 2.5 x 10 5 细胞/井在推荐的培养基中. </li>
	<li> 24 小时后, 使用倒置显微镜检查细胞, 同时使用10X 和40X 的目标来检测细胞形态学变化。如果需要, 记录照片. </li>
	<li> 使用 PBS 冲洗两次单元格。在37和 #176 中固定3.7% 甲醛中的细胞; C 在黑暗中孵化. 
	注: 甲醛固定在室温下工作也很好。在处理甲醛时, 要佩戴适当的个人防护设备. </li>
	<li> 在 PBS 中清洗单元格。将 ice-cold 甲醇中的细胞固定在 #8722; 20 和 #176; C 为15分钟 Permeabilize 的细胞, 在 3% BSA 和0.1% 海卫一在 pbs 冰上孵化他们10分钟, 用 pbs 将细胞洗净. </li>
	在室温下, <li> 在 PBS 溶液中孵育阻塞缓冲液 (3% BSA 和 0.1% Tween-20) 中的细胞. 
	注: 在4和 #176 孵化2小时; C 也很管用. </li>
	<li> 在 PBS 中准备 3% BSA 和 0.1% Tween-20 中的主抗体 (anti-tau 1:200) 解决方案 (, 如 、2和 #181; 0.4 mL 缓冲区中的 tau-13 抗体). </li>
	<li> 切割 pvdf 薄膜的小块, 使其在6孔板的每一个井内都能适当地适合; 浸泡在水中5分钟. 将切割的 pvdf 放在每个井中. </li>
	<li> 在每个井中添加60和 #181; 主要抗体解决方案。放置片, 使细胞能够触及主抗体溶液。在一个黑暗潮湿的室内过夜, 在4和 #176 孵育;PBS 四次清洗. </li>
	<li> 在 PBS 中制备二级抗体溶液 (荧光素-共轭 594 anti-mouse IgG, 1:400) 3% BSA 和 0.1% Tween-20. </li>
	<li> 切割 pvdf 薄膜的小块, 使其在6孔板的每一个井内都能正确地放置, 并将其浸泡在水中5分钟. 在每个井中放上 pvdf. </li>
	<li> 在 6-井板中添加80和 #181; 二级抗体溶液中的每一个 PVDF 片的 L。放置片使细胞能够接触二次抗体溶液。在一个阴暗潮湿的室温下孵育2小时, 用 PBS 洗涤四次. </li>
	<li> 在安装介质中装入4和 #39 的样品, 6-diamidino-2-吲 (DAPI), 并将片固定在玻璃幻灯片上. 
	<ol> <li> 在玻璃幻灯片上添加一滴安装介质和 DAPI。从每个井中取出片, 并将其放在含有 DAPI 的安装介质的玻璃幻灯片上。确保每个含有染色细胞的片的表面接触蔓在相应的玻璃滑梯上放上中号. </li>
		<li> 在每个片上涂抹指甲油, 密封并固定在玻璃上。如果需要, 请执行此步骤两次. </li>
	</ol> </li> <li> 在暗室中在室温下孵育1.5 小时. </li>
	<li> 在暗室中使用浸入式油对片进行共聚焦显微镜检查幻灯片. </li>
</ol>

作者声明他们没有什么可透露的。

该手稿建立了不同的程序条件, 检测总 tau 和磷酸化 tau 在大肠癌细胞治疗姜黄素和氯化。为了评估蛋白样品中 tau 的总磷酸化状态, 本文描述了磷酸酶的测定方法。这种检测方法有可能用于检测任何蛋白质的磷酸化状态。
这种方法是基于磷酸化蛋白比其 non-phosphorylated 状态慢的原理。本协议采用碱性磷酸酶和碱性磷酸酶缓冲剂。在将化验成分加入到细胞裂解后, 样品应在特定的...

头最初被确定为热稳定的微管相关蛋白, co-purified 与蛋白1。头是完全表达在更高的真核生物2,3,4。tau 的主要功能是控制微管组件1,5,6。它还有助于聚合的微管的7, 轴突传输的8, 轴突直径9, 形成的神经瘤极性, 和神经10。Tau 也充当蛋白质支架来控制一些信号通路。鼠脑研究表明, tau 是神经元特异的, 它主要本地化在轴突11。由于 tau 是微管聚合和神经元发育的关键, 因此 tau 被假设在中枢神经系统的轴突发育中起主要作用;这一假说后来通过体外和活体实验来验证。除了神经元, tau 是表达在不同的神经细胞细胞, 包括肝脏, 肾脏和肌肉细胞12,13。Tau 也被表达在人的乳房, 前列腺, 大肠, 胃, 和胰腺癌细胞系和组织14,15,16,17,18,19. 头也被发现在包涵体肌炎作为扭 tubulofilaments 在包含身体20。
头可进行几修饰修改。在所有修饰的修饰中, 磷酸化是最常见的。增加的 tau 磷酸化降低了它对微管的亲和性, 最终破坏了细胞骨架。八十五磷酸化部位已被描述在 tau 蛋白从人类阿尔茨海默病的脑组织分离。这些网站中, 53% 构成丝氨酸, 41% 苏氨酸, 只有6% 酪氨酸残留物21,22,23。Tau 磷酸化影响其定位, 功能, 约束力, 溶解度, 其易感性的其他修饰的修改。并且 tau 磷酸化到超过正常程度 (或完全饱和与磷酸盐小组) 被称为 tau, 复制结构和功能特征的阿尔茨海默氏病24。Tau 保持轴突微管的正常功能, 并确保在生理条件下的神经功能。然而, hyperphosphorylated tau 没有保持一个组织良好的微管结合, 导致神经元的损失, 因为微管的拆卸。tau 磷酸化的正常水平是需要正确的 tau 功能, 但 tau 无法正常工作, 如果其特征磷酸化水平被改变, 如果它是 hyperphosphorylated25。在阿尔茨海默氏病和其他一些与年龄相关的神经退行性疾病中, tau 成为 hyperphosphorylated, 形成配对的螺旋纤维和纤维缠结26,27。因此, 确定 tau 磷酸化和微管结合的方法很重要。
结直肠癌, 一个年龄相关的癌症, 是第三个最常见的诊断癌症和第三个突出的死亡导致的男性和女性的癌症28。大肠癌是西方世界主要的致死性癌症之一,29。因为结肠直肠癌和阿尔茨海默病都与衰老有关, 这两种疾病主要发生在发达国家, 那里的人们享有类似的饮食习惯, 这两个疾病可能会有某种程度的关联。此外, tau 阳性和 tau 阴性癌细胞对化疗药物的反应不同,例如, 紫杉醇16。
姜黄素是姜黄姜黄的主要衍生物之一, 印度香料姜黄30。几个世纪以来, 南亚人口每天都在饮食中消耗姜黄。姜黄素用于治疗不同的疾病, 包括结直肠癌, 阿尔茨海默氏病, 糖尿病, 囊性纤维化, 炎症肠病, 关节炎, 高血脂, 动脉粥样硬化和缺血性心脏病31,32,33,34,35,36,37,38. 锂也能杀死大肠癌细胞或防止其增殖39。锂也可用于治疗阿尔茨海默氏病40 , 因为它减少 tau 聚集, 并防止其 tau 在转基因鼠标模型41,42,43中观察到, 44。
本手稿旨在: 1) 测量治疗细胞的总 tau 和磷头表达水平;2) 描述磷酸酶的测定, 以测量整体 tau 磷酸化;3) 检查 tau 的微管结合;4) 用姜黄素或氯化在大肠癌细胞系中用共焦显微镜定位 tau。结果表明, 细胞治疗的姜黄素, 这是一个所谓的良好的化疗药物的结肠癌, 和治疗氯化可以减少在大肠癌细胞系的总 tau 和磷酸化 tau 的表达。这些处理也可能导致头的核易位。然而, 出乎意料的是, 姜黄素未能改善 tau 与微管的结合。

<table><tbody><tr><td>HCT 116 cell</td><td>ATCC</td><td>CCL-247</td><td></td></tr><tr><td>MEM (EBSS)</td><td>Hyclone</td><td>SH30024.01</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher (Gibco)</td><td>16000044</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>penicillin-streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>WelGene</td><td>LS 015-01</td><td></td></tr><tr><td>100 mm dish</td><td>Corning</td><td>430161</td><td></td></tr><tr><td>6 well plate</td><td>Corning</td><td>Coster 3516</td><td></td></tr><tr><td>Anti-Tau 13 antibody</td><td>abcam</td><td>ab19030</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Roche</td><td>10 708 984 001</td><td>Storage Temperature 2&amp;ndash;8 &amp;deg;C</td></tr><tr><td>Microlitre Centrifuges</td><td>Hettich Zentrifugen</td><td>MIKRO 200 R</td><td></td></tr><tr><td>Paclitaxel</td><td>Sigma-Aldrich</td><td>T1912</td><td>Storage Temperature 2&amp;ndash;8 &amp;deg;C</td></tr><tr><td>Curcumin</td><td>Sigma-Aldrich (Fluka)</td><td>78246</td><td>Storage Temperature 2&amp;ndash;8 &amp;deg;C</td></tr><tr><td>Microtubules (MT)</td><td>Cytoskeleton</td><td>MT001</td><td>Store at 4 &amp;deg;C (desiccated)</td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>Store at 4 &amp;deg;C in the dark</td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>GTP</td><td>Sigma-Aldrich</td><td>G8877</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>DPBS</td><td>WelGene</td><td>LB 001-02</td><td></td></tr><tr><td>Sonic Dismembrator</td><td>Fisher Scientific</td><td>Model 500</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100 XP</td><td></td></tr><tr><td>PIPES</td><td>Sigma</td><td>P1851</td><td></td></tr><tr><td>Bovine serum Albumin (BSA)</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Molecular Imager</td><td>Bio-Rad</td><td>ChemiDoc XRS+</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Protein assay dye reagent</td><td>Bio-Rad</td><td>500-0006</td><td></td></tr><tr><td>&amp;alpha;-tubulin (11H10) Rabbit mAb</td><td>Cell signalling</td><td>2125</td><td></td></tr><tr><td>GAPDH (14C10) Rabbit mAb</td><td>Cell signalling</td><td>2118</td><td></td></tr><tr><td>Anti-Tau (phospho S396) antibody</td><td>abcam</td><td>ab109390</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td>Store at room temperature</td></tr><tr><td>FastAP Thermosensitive Alkaline Phosphatase</td><td>Thermo Scientific</td><td>EF0651</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td>Store at room temperature</td></tr><tr><td>Phosphatase Inhibitor Cocktail</td><td>Cell Signalling</td><td>5870</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Cell Signalling</td><td>5871</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>RIPA Buffer</td><td>Sigma</td><td>R 0278</td><td>Storage Temperature 2&amp;ndash;8 &amp;deg;C</td></tr><tr><td>Tau-352 human</td><td>Sigma</td><td>T 9950</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X - 100</td><td>Store at around 25 &amp;deg;C</td></tr><tr><td>PVDF membrane</td><td>Bio-Rad</td><td>162-0177</td><td></td></tr><tr><td>Goat anti-mouse IgG Secondary Antibody</td><td>ThermoFisher</td><td>A-11005</td><td>Store at 4 &amp;deg;C in the dark</td></tr><tr><td>Confocal Microscopy</td><td>Leica Microsystem</td><td>Leica TCS SP5</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix</td><td>75819</td><td></td></tr><tr><td>Protein Assay</td><td>Bio-Rad</td><td>500-0006</td><td>Store at 4 &amp;deg;C</td></tr></tbody></table>

assay for phosphorylation and microtubule binding along with localization of tau protein in colorectal cancer cells

微管相关蛋白 tau 是一种神经元蛋白, 本地化主要在轴突。一般来说, tau 是正常的神经功能的必要条件, 因为它涉及微管组装和稳定。除了神经元, tau 是在人的乳房, 前列腺, 胃, 结直肠癌和胰腺癌, 它显示几乎类似的结构和发挥类似功能的神经元 tau。tau 及其磷酸化的数量可以改变其作为微管的稳定剂的功能, 并导致在不同的神经退行性疾病, 如阿尔茨海默氏病的配对螺旋纤维的发展。确定 tau 的磷酸化状态及其微管结合特性是很重要的。此外, 检查 tau 的细胞内定位是重要的不同疾病。这份手稿详细的标准协议, 测量 tau 磷酸化和 tau 结合到微管的大肠癌细胞与或没有姜黄素和氯化治疗。这些治疗可以用来阻止癌细胞的增殖和发育。在使用低剂量的抗体时, 使用免疫组化和共聚焦显微镜检查 tau 的细胞内定位。这些化验可以反复用于筛选影响 tau tau 或微管结合的化合物。新的治疗方法用于不同的 tauopathies 或相关的抗癌药物, 可以潜在的特点使用这些协议。

用不同浓度的姜黄素或氯化 (图 1) 治疗后, 对全头和磷的表达进行了研究。三种不同浓度的姜黄素对细胞的治疗降低了 tau 表达水平;但是, 磷在低浓度姜黄素的治疗下增加, 但在处理姜黄素浓度较高的细胞时却减少。抗磷头 (Ser396) 用于检测磷头。在三种不同浓度的氯化 (图 1) 中, 总 tau 和磷的水平都降低了。先前的研究表明, tau ?...

Watch this Scientific Journal Video about Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells at JoVE.com

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

本手稿描述了测量 tau tau, 测量 tau 结合到微管的标准协议, 以及药物治疗后细胞内 tau 的定位。这些协议可以重复使用, 以筛选药物或其他化合物的目标 tau 或微管结合。

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

assay-for-phosphorylation-microtubule-binding-along-with-localization

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.9K 次观看.  KIST Gangneung Institute of Natural Products. 本手稿描述了测量 tau tau, 测量 tau 结合到微管的标准协议, 以及药物治疗后细胞内 tau 的定位。这些协议可以重复使用, 以筛选药物或其他化合物的目标 tau 或微管结合。

视频: Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

科研

生物化学

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

陶子细胞定位作为研究疾病相关基因表达的工具的调制

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

遗传学

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

生物工程

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

神经科学

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

微胶质细胞抗体介导的陶氏清除研究

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

生物学

Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Source: Danis, C. et. al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55001">Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.

核磁共振波谱鉴定蛋白质中的多种磷酸化

Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered ...

JoVE Encyclopedia of Experiments

Biological Techniques

Spectroscopy Techniques

A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

Source: De Marco, D. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58576">Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.</a>&#160;J. Vis. Exp. (2018)
This video showcases a cell-based assay quantifying Tau uptake by murine microglia using pH-sensitive fluorescent-labeled Tau aggregates, analyzed via fluorescence-activated cell sorting for a quantitative assessment.

一种基于细胞的测定法，用于评估小胶质细胞对 tau 蛋白的摄取

1. BV-2 Microglia Cells Culture
NOTE: Handle BV-2 cells under Biosafety Level 2 containment. The BV-2 cell line produces an enveloped recombinant ...

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding Tau Protein in Colorectal Cancer Cells

Source: Huda, M. N., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/55932"> Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells.</a> J. Vis. Exp. (2017).
This video describes the microtubule-binding assay used to separate the microtubule-binding tau protein from non-binding hyperphosphorylated tau protein fractions in colorectal cancer cells. This assay helps determine the effect of various chemicals that can alter the phosphorylation status of tau protein, affecting its microtubule binding.

CRC 细胞中的微管结合测定:一种检查结直肠癌细胞中微管结合 tau 蛋白的方法

Source: Huda, M. N., et al.  Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells. J. Vis. ...

癌症研究

Colorectal Cancer

Assays & techniques

Phosphatase Assay in CRC Cells: A Method to Examine the Phosphorylation Status of Tau Protein in Colorectal Cancer Cells

Source: Huda, M. N., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/55932"> Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells.</a> J. Vis. Exp. (2017)
This video describes a protocol for assessing the phosphorylation status of tau protein by a phosphatase assay in colorectal cancer cells. Phosphatase enzyme acts on the phosphate groups present on the tau protein, increasing the mobility of tau protein on SDS PAGE gel compared to the untreated tau protein samples.