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, 样品应在特定的温度下在特定的最佳时间内孵化。在孵化后, 样品应在 SDS 取样缓冲液中煮沸, 以停止反应。以前, 不同的实验协议可能被用来评估 tau 与微管的结合。在这里提出的微管结合试验很容易执行, 同时包括积极和消极的控制, 以提供质量保证使用上清和颗粒分数。mt 唯一的阴性控制被用来验证 mt 不包含任何束缚内生 tau。此外, 纯人类 tau-352, 一个微管结合头被用作阳性对照。用非约束分数 (上清) ultra-speed 离心分离微管结合分数 (颗粒);此过程可能是一个限制, 因为这样的 ultracentrifuge 是昂贵的, 而且没有广泛的可用。此外, 由于使用了少量的样品, 在 ultracentrifuge 转子中需要使用带有适配器的细长离心管。虽然可以重复使用这种管子, 但使用一次性是可取的, 以避免交叉污染。最后, tau 定位协议的优点是只需要少量的初级和次级抗体。在固定、性和阻断附着于片的细胞后, 一小滴抗体沉积在一小块 PVDF 上, 然后放在一个6孔板的井里。片被保留, 以便抗体能进入细胞。通过使用该协议, 可以最小化所使用的抗体量。
应牢记一些预防措施, 以确保可靠和可量化的结果。首先, 严格的无菌技术应用于细胞培养和细胞提取物的污染。完整的帕裂解缓冲液应准备新鲜。在磷酸酯酶测定中, 在孵育磷酸酶治疗和未经处理的样品后立即将样品煮沸3分钟并开始 SDS-page 是很重要的。Tau-352 应分为小等分, 并保持在冰后, 从20° c 的冷冻库, 并立即存入冰箱后使用后。从−80° c 去除后, 如果在72小时内再使用, MT 储存应保持在冰上;否则, 保持室温。PEM 缓冲区应准备作为一个10x 的浓缩解决方案, 因为1x 缓冲区需要新鲜作出时, GTP 和紫杉醇添加。
Tau 函数受其磷酸化程度的调节。tau tau 不发生在正常成人脑 tau 类似 tau-352, 这是作为积极控制的微管结合检测。然而, tau tau 发生在神经退行性疾病。该协议和随后的结果表明, 人大肠癌细胞线也携带磷酸化 tau 类似阿尔茨海默氏病的大脑。使用大剂量姜黄素, 特别是氯化可以减少 tau 磷酸化。因此, tau 的神经退行性疾病, 如阿尔茨海默氏病和结直肠癌可能存在良好的相关性, 而姜黄素或氯化可用于治疗结直肠癌和阿尔茨海默病。最后, 研究 tau 磷酸化和 tau 微管结合不仅对神经退行性疾病, 而且对某些化疗有重要意义, 因为改变 tau 微管结合的药物是动态研究的癌症疗法51。在这里提出的协议将有可能有助于发现和发展新的治疗药物治疗不同的 tauopathies 和癌症。

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 &#176;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&#8211;8 &#176;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&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Curcumin</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>78246</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Microtubules (MT)</td>
			<td>Cytoskeleton</td>
			<td>MT001</td>
			<td>Store at 4 &#176;C (desiccated)</td>
		</tr>
		<tr>
			<td>Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Store at 4 &#176;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 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Protein assay dye reagent</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-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 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Cell Signalling</td>
			<td>5871</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Sigma</td>
			<td>R 0278</td>
			<td>Storage Temperature 2&#8211;8 &#176;C</td>
		</tr>
		<tr>
			<td>Tau-352 human</td>
			<td>Sigma</td>
			<td>T 9950</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>X - 100</td>
			<td>Store at around 25 &#176;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 &#176;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 &#176;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 的表达水平在不同的癌症类型和不同的癌症类型部位各不相同。以前关于结直肠癌的数据显示, tau 是用两个细胞线 (小贩116和 SW480) 来表达的, 而结直肠癌也有磷酸化19。用5µM 姜黄素处理的细胞中磷的 tau 水平高于未治疗的细胞。在相同浓度的姜黄素处理的细胞中, 磷的 tau 水平高于总 tau。10µM 姜黄素治疗细胞与未处理的细胞相比, 磷 tau 水平降低, 但磷的表达仍高于总 tau 表达水平。治疗细胞与30µM 姜黄素降低磷 tau 表达与磷 tau 在未经处理的细胞和总 tau 水平在相同的治疗细胞。
这份手稿建立了一个简单的协议, 以评估磷酸化状况的 tau 磷酸酶检测 (图 2)。在姜黄素治疗后, 整体 tau 磷酸化没有明显变化, 在特定氨基酸残留物上磷酸化显著 (图 1)。与未处理的细胞相比, 氯化治疗的细胞中总的 tau 磷酸化停止。磷酸酶处理的样品比未经处理的样品电泳快, 验证未经治疗的样品比磷酸酶治疗的样品更 hyperphosphorylated。姜黄素治疗的细胞显示几乎相同的结果, 表明治疗大肠癌细胞系没有减少 tau 磷酸化, 如图 1所示。磷酸酶治疗和未经处理的样品电泳在与氯化治疗的细胞样本中几乎相同的范围内, 这表明这些细胞中的 tau 磷酸化减少了 (图 2)。在这里, 高浓度 (20 µM 或30µM) 的姜黄素处理的样本, 以比较整体磷酸化状态, 低浓度处理显示更高 site-specific 磷酸化显示的特定磷头 (S396) 抗体。
此外, 在这个实验室中, 成功地建立了细胞样品的微管结合分析, 使用 tau-352 作为一个正控制, 只有 MT 作为负控制 (图 3)。对于姜黄素和氯化的治疗, 微管结合活性被抑制, 通过微管结合试验证明。在本实验中, 姜黄素的治疗没有显示微管结合能力, 但抑制了类似于以前的研究46,47的绑定, 而它是有效的抑制 site-specific tau 磷酸化在浓度较高。在图 1中, 10 µM 姜黄素的治疗结果与对照相比, 磷的表达极少, 但表达比总 tau 高。然而, 在未经处理的样品中, 氯化治疗后大肠癌头的微管结合能力降低, 且低浓度的小管。特定部位的 tau 磷酸化效应微管结合和 self-aggregation48。富脯氨酸区域的 Tau 磷酸化阻碍了微管结合特性, 而 c-端区则增加了这些特性, 而这两个区域随着 MT 结合区的结合性能降低了约 70%, 并使微管48。其他一些因素, 以及其他 site-specific 磷酸化的 tau 可能涉及这种微管不稳定的大肠癌 tau 治疗与姜黄素或氯化。
一个简单的协议使用少量的初级和二级抗体, 使 tau 在大肠癌细胞系的定位后, 姜黄素治疗 (图 4)。结果表明, 在姜黄素治疗后, tau 移位到细胞核, 这一发现类似于早期的研究报告, 核 tau 是神经退行性疾病如阿尔茨海默氏病等神经元 DNA 保护的关键参与者49 ,50。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55932/55932fig1.jpg"> 
图 1: 在姜黄素或氯化治疗后的大肠癌细胞中 tau 和磷头的表达.用三种不同浓度的 (A) 姜黄素和 (B) 氯化在10% 聚丙烯酰胺凝胶上, 用 SDS-page 对 24 h 处理过的对照细胞或细胞提取样品, 并用 anti-tau 或 anti-phospho 抗体进行了探讨。在右面板上, 对印迹结果进行了密度分析。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55932/55932fig1large.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/55932/55932fig2.jpg"> 
图 2: 在未经治疗或差异处理的细胞样品中, 用磷酸酯酶法检测 tau 的整体磷酸化.奇数车道显示控制细胞提取物, 而甚至车道显示磷酸酶处理的姜黄素处理或氯化处理的样品。磷酸酶的治疗使 tau 电泳移动速度比未经处理的样品, 其中含有磷酸化 tau。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55932/55932fig2large.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/55932/55932fig3.jpg"> 
图 3: 通过微管结合试验检测到的大肠癌细胞中的 tau 细胞结合.控制小贩116细胞样本, 姜黄素治疗, 或氯化处理的细胞样本和大肠杆菌头被孵化的微管, 详见协议4节。immunoblotted 与 anti-tau 抗体的相同数量的上清 (S) 和颗粒 (P) 分数。仅含 mt 的负控制车道运行, 以确认 mt 没有包含任何约束内生 tau。下面板显示了对单个样品的西方印迹的密度分析, 使上清 (未粘合头) 和颗粒 (束缚 tau) 分数的比较。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55932/55932fig3large.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/55932/55932fig4.jpg"> 
图 4: 用共焦麦克风检测 Tau 蛋白的定位roscopy Tau 在大肠癌细胞中的细胞核被局部化。左面板显示头定位检测使用 anti-tau 单克隆抗体, 而中间板显示核染色的 DAPI。有代表性的例子, 显示头易位进入细胞核的处理细胞也显示。控制细胞主要表现在细胞核周围和外侧, 而在被处理的细胞中, tau 也会在细胞核内定位。缩放栏 = 20 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55932/55932fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

科研

生物化学

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...