我们感谢 Dr. 武 Iwatsubo (日本东京大学) 提供的质粒编码 3 xflag-LRRK2 重量和突变体。我们还感谢 Dr. 达里奥阿莱西 (英国邓迪大学) 提供 MLi-2 和质粒编码 HA-Rab10。这项工作得到了日本促进科学协会 (jsp) KAKENHI 赠款号 JP17K08265 (gi) 的支持。

1. P 标记 SDS–PAGE 的样品制备
<ol>
	<li>从10厘米的盘子中取出并丢弃培养基, 其中细胞是用5毫升 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 的吸入和洗涤细胞生长的, 首先在盘子的一侧加入 DPBS, 以避免干扰细胞层, 然后手工把盘子放回来回几次。</li>
	<li>去除和丢弃 DPBS 使用吸入和添加2毫升的 0.25% (瓦特/v) 胰蛋白酶稀释 DPBS, 并轻轻地岩石的盘子覆盖的细胞层。将菜肴放入 co2孵化器 (37 ° c, 加湿空气, 5% co2) 为5分钟。</li>
	<li>移后使用一次性吸管分解分离细胞, 收集细胞悬浮到一个15毫升管和测量的细胞密度使用 hematocytometer 在显微镜下18。</li>
	<li>将细胞稀释到 2.5 x 105细胞/毫升与 Dulbecco 的改良鹰培养基 (DMEM) 补充 10% (v/五) 胎牛血清 (FBS), 100 U/毫升青霉素, 100 µg/毫升链霉素。添加2毫升 (5 x 105细胞) 的稀释细胞悬浮每井 6-井板。</li>
	<li>在一个 co2孵化器 (37 ° c, 湿空气, 5% co2) 过夜的细胞生长。</li>
	<li>转染 HEK293 细胞 注意: 如果要检查内源性 Rab10 的磷酸化, 请继续步骤1.7。本协议中使用的质粒可根据要求从作者那里获得。有关其 DNA 序列的简要信息和图 S1 , 请参见材料表。<ol>
		<li>分200µL DMEM 成1.5 毫升的管子。</li>
		<li>对每管, 添加0.266 µg (最终浓度1.33 µg/毫升) 的质粒编码 HA-Rab10 和1.066 µg (最终浓度5.33 µg/毫升) 的质粒编码 3×FLAG LRRK2 从500µg/毫升质粒股票。然后加入4µL 1 毫克/毫升溶液的聚乙烯 (溶解在20毫米 HEPES-氢氧化钠, pH 7.0), 并立即混合解决方案由涡流 5 s。</li>
		<li>让管站在室温下10分钟, 并增加一个管滴的内容使用微。轻轻地和手工晃动的文化板块来回几次, 让转染混合均匀弥漫在整个井。</li>
	</ol>
	</li>
	<li>在 co2孵化器 (37 ° c, 湿空气, 5% co2) 中, 让细胞在另外的24-36 小时内生长。</li>
	<li>如果内源性 Rab10 磷酸化将被审查, 治疗细胞与和没有 LRRK2 抑制剂为 1 h 在溶细胞之前。<ol>
		<li>在10毫米的二甲基亚砜 (亚砜) 中溶解抑制剂, 制备 LRRK2 抑制剂的库存溶液。我们建议使用 MLi-2 和 GSK2578215A, 这是高度特异和有效的 LRRK2 抑制剂。库存解决方案在-80 ° c。</li>
		<li>通过用亚甲基亚砜稀释股票溶液来制备抑制剂的工作库存: 为 MLi-2, 分别为内生和表达 LRRK2 准备10µM 和30µM。对于 GSK2578215A, 分别为内生和表达 LRRK2 准备1毫米和3毫米。</li>
		<li>添加2µL 的工作库存 MLi-2 或 GSK2578215A 到中间的一个井使用微和轻轻晃动板的来回几次, 让抑制剂扩散均匀整个井。</li>
		<li>将2µL 的亚甲基亚砜添加到不同的井中, 以类似的方式对1.8.3 中的抑制剂进行负控制。</li>
		<li>把盘子放回孵化室, 培养1小时的细胞。</li>
	</ol>
	</li>
	<li>溶解细胞。<ol>
		<li>把文化板放在冰上。删除并丢弃媒体。先将2毫升的 DPBS 到盘子的一侧, 洗净细胞, 避免干扰细胞层, 并多次手工地将盘子来回摇动。</li>
		<li>去除和丢弃 DPBS, 并添加到细胞100µL 的裂解缓冲液 (50 毫米三盐酸 pH 7.5, 1% (v/v) 聚氧乙烯 (10) 苯基醚, 1 毫米 EGTA (乙二醇-双 (2-aminoethylether)-n, n, n, n-乙酸酸), 1 毫米钠钒, 50 毫米氟化钠, 10 毫米β甘油, 5 毫米焦磷酸钠, 0.1 µg/毫升微囊藻毒素-LR, 270 毫米蔗糖, 蛋白酶抑制剂鸡尾酒)。</li>
		<li>在冰上倾斜盘子, 用刮板机刮掉细胞, 尽可能地收集尽可能多的细胞。收集裂解使用微到1.5 毫升管 (pre-chilled 冰)。 警告: 微囊藻毒素-LR 可能是致命的, 如果吞食或与皮肤接触。</li>
	</ol>
	</li>
	<li>让管子在冰上站立10分钟完全溶解。用离心法 (2万 x g, 10 分钟, 4 ° c) 裂解, 将清转移到新的1.5 毫升 pre-chilled 冰上。</li>
	<li>用布拉德福法测定清除裂解中的蛋白质浓度 (µg/µL)。<ol>
		<li>用蒸馏水稀释储存液, 制备牛血清白蛋白 (BSA) 标准 (0.2、0.4、0.6、0.8 和1毫克/毫升)。用蒸馏水稀释20倍的清除细胞裂解。</li>
		<li>将 BSA 标准的5µL/井, 空白 (蒸馏水), 和每一个稀释的细胞裂解成一个 96-井板一式三份。</li>
		<li>使用12通道微加150µL/井的布拉德福德试剂, 让板在室温下站立5分钟。</li>
		<li>测量平板阅读器上 595 nm 的吸光度, 并与 BSA 标准进行比较。</li>
	</ol>
	</li>
	<li>为 SDS 页准备100µL 样品。样品的蛋白质浓度为1µg/µL 为表达 HA-Rab10 和2µg/µL 为内生 Rab10。<ol>
		<li>利用步骤1.11.4 的量化浓度, 计算表达 (HA-Rab10) 的蛋白质浓度, 将蛋白质量 (100 µg 或 200 Rab10) 除以蛋白含量 (µg) 或200µg (内源性裂解) 的裂解 (µL) 的体积 (µg)。克/µL)。</li>
		<li>添加25µL 的 4x Laemmli 的 SDS 页样本缓冲区 (62.5 毫米三盐酸, pH 值 6.8, 8% (w/v) SDS, 40% (v/五) 甘油, 0.02% (溴) 蓝色, 4% (v/v) β-基) 到新的1.5 毫升管保持室温。</li>
		<li>在室温下, 将单元格裂解的计算量添加到每个管中, 并由涡流 5 s 混合。</li>
		<li>将总体积100µL 与裂解缓冲液混合, 在室温下涡流5。</li>
	</ol>
	</li>
	<li>补充与10毫米 MnCl2 , 以淬火螯合剂。添加1µL 500 mM MnCl2。在室温下由涡流 5 s 混合。 注: 包含 MnCl2的样本可能不适用于普通 SDS 页。</li></ol>这一步是由于螯合剂 (如乙酸酸 (EDTA)、EGTA、等) 在裂解缓冲液中的存在而需要的。否则, 锰2 +离子耦合到 p 标记丙烯酰胺将被螯合剂分离, p 标记 SDS 页将无法正常工作。
	<li>将所有样品在100° c 下煮沸5分钟, 并将样品储存在-20 ° c 以下直到使用。煮沸的样品可以被存放至少6月。</li>

2. 用于 p-标签 SDS–PAGE 的铸造凝胶
注: 凝胶应在运行凝胶的同一天进行。凝胶可以在环境光照条件下进行。
<ol>
	<li>准备5毫米 p 标记丙烯酰胺库存溶液, 首先将10毫克的粉末/固体 p 标记丙烯酰胺完全溶解, 再用100µL 甲醇, 然后通过添加双蒸馏水将其带到3.3 毫升。 注: P 标签丙烯酰胺是轻敏感。准备好的溶液应贮存在4° c 至使用的黑暗中。</li>
	<li>通过喷洒70% 乙醇, 用纸巾擦净和缺口板。把凝胶板组装起来。在这个特定的协议中使用的凝胶板的尺寸是80或100毫米长100毫米宽。干净的硅垫片放在普通和缺口板之间, 组装的板材用粘合剂夹夹住。 注: 可用于普通 SDS 页的任何类型的凝胶板。</li>
	<li>把梳子用于 (17 井的塑料梳) 放入凝胶板和标记在凝胶板材底部的位置井用永久标记。</li>
	<li>准备10毫升的10% 丙烯酰胺凝胶混合物 (10% (w/v) 丙烯酰胺 (丙烯酰胺: 双-丙烯酰胺 = 29:1), 375 毫米三盐酸 (pH 8.8), 0.1% (瓦特/v) SDS) 在15毫升管。 注: 丙烯酰胺的最佳浓度可能因所使用的试剂而异。此时不应添加 Tetramethylethylenediamine (TEMED) 和过硫酸铵 (APS)。</li>
	<li>添加100µL 5 毫米 P 标记丙烯酰胺和10µL 1 M MnCl2解决方案, 最终浓度分别为50µM 和100µM。 注: P 标记丙烯酰胺和 MnCl 的最佳浓度2也可能因所使用的试剂而异。</li>
	<li>在最后浓度为 0.15% (v/v) 的凝胶混合物中加入15µL 的 TEMED, 然后50µL 10% (w/v) APS, 最终浓度为 0.05% (w/v)。混合好, 轻轻旋转管 5 s, 并倒入组装板, 立即高达2毫米以下的位置在步骤2.3 标记。</li>
	<li>轻轻地将 2-丙醇层放到凝胶溶液上, 使分离凝胶的顶端扁平化。</li>
	<li>让凝胶在室温下站立30分钟。没有必要保护凝胶免受光照。 注: 凝胶在室温下设置可能需要较长的时间。在加入 TEMED 和 APS 之前, 先脱去凝胶混合物有助于加速这一步。为此, 凝胶混合物可以在100-200 毫升的锥形烧瓶中进行准备, 连接到一个吸力。德加的解决方案为10分钟。</li>
	<li>用纸巾将 2-丙醇去除。</li>
	<li>洗净凝胶的顶部空间, 用蒸馏水从洗涤瓶中填满空间, 然后倒入盆中丢弃水。重复洗涤3次。</li>
	<li>用纸巾把残留在凝胶顶部的剩余水分去除。</li>
	<li>准备3毫升的4% 丙烯酰胺凝胶混合物 (4% (w/v) 丙烯酰胺 (a: crylamide: 双-丙烯酰胺 = 29:1), 125 毫米三盐酸 (pH 6.8), 0.1% (瓦特/v) SDS) 在15毫升管。 注: 请勿将 P 标记丙烯酰胺或 MnCl2溶液添加到堆积凝胶混合物中。</li>
	<li>在最后浓度为 0.25% (v/v) 和 0.08% (w/v), 分别加入7.5 µL TEMED 和24µL 10% (w/v) APS。混合好, 轻轻旋转管 5 s 和倒入混合物顶部的分离凝胶。立即放适当的梳子 (例如17井塑料梳子, 可容纳25µL 的样品)。</li>
	<li>让凝胶在室温下站立30分钟。没有必要保护凝胶免受光照。堆叠凝胶设置后, 运行他们没有进一步的存储。</li>
</ol>
3. SDS–PAGE 和免疫
<ol>
	<li>从凝胶中取出梳子。然后取下硅垫片, 然后从凝胶中取出夹子。</li>
	<li>将浇铸的凝胶放入凝胶罐中, 通过夹紧夹子将凝胶固定在罐内。</li>
	<li>将运行的缓冲液 (25 毫米三, 192 毫米甘氨酸, 0.1% (w/v) SDS) 倒在凝胶的底部和顶部。清洁井通过冲洗运行缓冲区使用5毫升注射器和21G 针, 以消除凝胶件。</li>
	<li>使用附有注射器的弯曲针头从凝胶底部的空间中除去气泡。要做一个弯曲的针, 手动弯曲针中间的21G 针, 使针尖和针的底部之间的角度成为30-45 °。</li>
	<li>旋转下来的沉淀物造成的 MnCl2在 2万 x g 在室温下1分钟, 以获得明确的样品。</li>
	<li>负载10µg 蛋白检测表达 Rab10 和30µg 蛋白的磷酸化的内源性 Rab10。 注: 在所有井中加载等量的样品是至关重要的。空车道应加载 1x Laemmli 的 SDS 页样本缓冲区。如果示例包含 MnCl2, 则将相同浓度的 MnCl2添加到虚拟示例中。</li>
	<li>用分子量标记 (MWM) 装载的井也应辅以 1x Laemmli 的 SDS 页样本缓冲器, 以便在所有井中加载的样品量相同。 注意: 同样, 如果示例包含 MnCl2, 则将相同浓度的 MnCl2添加到 MWM。另外, 可以使用无 EDTA 的 MWM。</li>
	<li>运行凝胶在 50 V 为堆积 (大约30分钟) 直到染料前面十字架入分离胶凝体。</li>
	<li>在样品堆之后, 将电压改为 120 V, 直到染料前部到达凝胶的底部 (分别为80和 100 mm 长凝胶的大约50和80分钟)。 注: MWM 的迁移模式与正常 SDS 页凝胶的移植方式不同。它不应用于估计 p 标记 SDS-页凝胶中蛋白质的分子量, 但可用于检查 p 标记凝胶的重现性。有关详细信息, 请参阅讨论。</li>
	<li>清洗凝胶, 从凝胶中去除 MnCl2 。<ol>
		<li>将转移缓冲液 (48 毫米三, 39 毫米甘氨酸, 20% (v/五) 甲醇) 倒入含有10毫米 EDTA 和 0.05% (w/v) SDS 的容器 (例如, 大型称重船)。</li></ol></li></ol>缓冲液的体积应足以覆盖凝胶。
		<li>从凝胶板上取出分离凝胶, 并在一个容器中放入一个凝胶。丢弃堆积凝胶。</li>
		<li>在室温下, 将凝胶放在摇摆振动筛 (60 rpm) 上10分钟。</li>
		<li>重复洗涤步骤共3次。 注: 每次洗涤使用新的缓冲。</li>
		<li>用含有 0.05% (w/v) SDS 的转移缓冲器清洗一次10分钟的凝胶以除去 EDTA。缓冲液的体积应足以覆盖凝胶。</li>
	
	
	<li>电转移到硝化纤维或聚偏氟 (PVDF) 膜使用湿罐。<ol>
		<li>将滤纸 (10 x 7 cm) 放在海绵垫上进行传输。将凝胶放在滤纸上。确保过滤纸和凝胶之间没有气泡。</li>
		<li>在凝胶上放一个膜 (10 x 7 厘米), 确保凝胶和膜之间没有气泡。</li>
		<li>将另一张滤纸 (10 x 7 厘米) 放在膜上, 并再次确保膜和滤纸之间没有气泡。</li>
		<li>将另一个海绵垫放在滤纸上。将膜/滤纸叠放在盒式磁带中进行传输。</li>
		<li>将卡带放入转印槽中, 确保膜位于凝胶和正极带电的阳极之间。</li>
		<li>将油箱连接到电源, 并将油箱放入装有 ice-cold 水的苯乙烯泡沫箱中。开始转移在 100 V 为180分钟。 注: 长期转移是必要的, 因为从 P 标记的 sds-页凝胶蛋白的转移不像普通 sds 页凝胶那样有效。有效的冷却是必不可少的, 以避免融化凝胶在转移。</li>
	</ol>
	</li>
	<li>检查转移
	<ol>
		<li>用镊子将膜从凝胶中取出, 浸泡在胭脂红 s 溶液中 (0.1% (w/v) 胭脂红 s, 5% (v/v) 乙酸), 以在塑料容器中将转移的蛋白质染色。溶液的体积应足以覆盖膜。</li>
		<li>通过手动摇动在室温下孵育1分钟的膜。 注意: 每条车道上都应该有一个带阶梯。</li>
		<li>用镊子从染色液中挑出膜, 看看带上样品的每一条车道上的阶梯是否都有均匀的花纹和强度。</li>
		<li>检查染色后, 取出染色液, 并添加 TBST 缓冲 (20 毫米三盐酸盐 pH 7.4, 150 毫米氯化钠, 0.1% (v/v) polyoxyethylenesorbitan 酸)。缓冲区的体积应足以覆盖膜。 注意: 胭脂红的解决方案可以收集和重复多次。</li>
		<li>在室温下, 在 TBST 上摇动摇床 (60 rpm) 的细胞膜, 直到膜上没有可见的条纹。</li>
		<li>用新鲜的 TBST 重复洗涤步骤5分钟。</li>
	</ol>
	</li>
	<li>删除并丢弃 TBST。通过加入 5% (w/v) 脱脂牛奶溶解在 TBST 和摇摆在一个振动筛 (〜 60 rpm) 在室温下1小时。堵塞溶液的体积应足以覆盖膜。</li>
	<li>通过稀释原抗体 (anti-Rab10 抗体的内源性 Rab10 和抗 HA 抗体表达 HA-Rab10) 在10毫升每膜的阻断溶液 (参见材料表的浓度)。</li>
	<li>移除并丢弃阻塞解决方案并添加主抗体。在4° c 过夜的摇摆振动筛 (60 rpm) 上孵育膜。</li>
	<li>去掉主抗体溶液, 加入 TBST 洗膜。缓冲区的体积应足以覆盖膜。</li>
	<li>在室温下, 在摇摆振动筛 (60 rpm) 上孵育5分钟的膜。每次使用新鲜 TBST 重复洗涤 (步骤 3.16) 总共3次。</li>
	<li>用辣根过氧化物酶 (HRP) 在每膜10毫升的阻断溶液中稀释二次抗体, 制备二次抗体溶液。用 hrp anti-Rab10 抗体标记的兔 igg 抗体, 用 hrp 标记的 anti-mouse igg 抗体对抗 HA 抗体进行检测 (见材料表进行浓缩)。</li>
	<li>在第三次洗涤后除去并丢弃 TBST, 并加入二次抗体溶液。在室温下, 在摇动振动筛 (60 rpm) 上孵育1小时的膜。</li>
	<li>在 TBST 洗膜10分钟, 重复洗涤步骤总共3次, 类似于步骤3.17。</li>
	<li>利用增强的化学发光 (ECL) 开发膜。 注意: 曝光时间可能因 ECL 溶液和用于检测化学发光的系统而异。<ol>
		<li>打开一个装有电荷耦合器件 (CCD) 摄像机和连接到成像器的计算机的成像仪。启动成像仪的控制软件。等到 CCD 摄像机的温度达到-25 ° c。</li>
		<li>把1毫升的 ECL 解决方案的一个膜上的塑料包装上蔓延的长凳上。</li>
		<li>在 ECL 溶液上放一个膜凝胶煎, 然后快速翻转, 使膜的两面都涂上溶液。</li>
		<li>通过让膜的一面触摸纸巾5秒来把膜吸干并排出。</li>
		<li>把膜放在清晰的胶片 (例如, 清晰的纸袋) 之间。 注: 不建议包装膜的塑料包装。用于包装膜的透明膜需要尽可能平整, 避免出现明显的皱纹, 以免产生不均匀的背景。</li>
		<li>将膜放在黑色托盘上。把托盘放在成像器里, 关上门。</li>
		<li>单击控制软件窗口中的 "聚焦" 按钮。检查膜是否正确定位。单击 "返回" 按钮。</li>
		<li>为 "曝光类型" 选择 "精度"。为 "曝光时间" 选择 "手动", 并将曝光时间设置为1秒。</li>
		<li>为 "灵敏度/分辨率" 选择 "高"。保留 "添加数字化图像" 未选中。单击 "开始" 按钮以选择图像。</li>
		<li>将计算机上显示的图像保存为 TIFF 文件。</li>
		<li>重复拍摄的曝光时间从 1, 3, 10, 30, 60, 90, 120, 150s 和高达180s。在拍摄最后一幅图像时, 要检查 "添加数字化图像", 以使该膜的数字图像, 而不是化学发光, 可以同时采取。</li>
		<li>选择具有最大动态范围的最佳图像, 并在感兴趣的波段中不含任何饱和像素 (以红色显示)。 注: 常规 X 射线胶片也可用于检测19。</li>
	</ol>
	</li>

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作者没有什么可透露的。

在这里, 我们描述了一个简便和稳健的方法检测 Rab10 磷酸化的 LRRK2 在内生水平的基础上, P 标记方法。由于目前对磷酸化 Rab10 的抗体仅适用于表达蛋白15, 目前采用 p-标记的方法是评价 Rab10 磷酸化的内源水平的唯一途径。此外, 本方法还允许估计细胞中 Rab10 磷酸化的化学计量。因为 P 标记方法一般适用于磷-蛋白质, 所以本协议可以是为其他磷蛋白的相似方法建立的 "原型"。
<p ...

PD 是最常见的神经退行性疾病之一, 主要影响多巴胺能神经元在中脑, 导致功能障碍的老年人的马达系统的1。虽然大多数病人是以零星的方式发展 PD, 但仍有家庭继承这种疾病。一些基因的突变被发现与平板的2相联系。平板的致病基因之一是 LRRK2, 其中八义突变 (N1437H, R1441C/G/H/S, Y1699C, G2019S, I2020T) 链接到一个主导的遗传的平板被称为 PARK8 迄今已被报告3,4,5。数个全基因组的联合研究 (GWAS) 的零星 pd 患者也确定了 LRRK2 位点的基因组变异作为帕金森病的危险因素, 这表明 LRRK2 的功能异常是神经的常见原因。和 PARK8 平板显示器6,7,8。
LRRK2 是一种大蛋白质 (2527 氨基酸), 由富含亮氨酸的重复域、GTP 复合蛋白 (roc) 域的结合 Ras、roc (林) 域 C 端、丝氨酸/苏氨酸蛋白激酶域和 WD40 重复域9组成。八平板的突变定位在这些功能领域;N1437H 和 R1441C/G/H/S 在 ROC 域, Y1699C 在林域, G2019S 和 I2020T 在激酶领域。自 G2019S 突变, 这是最常见的突变在 PD 患者10,11,12, 增加了激酶活性的 LRRK2 2-3 倍在体外13, 这是假设LRRK2 基质磷酸化的异常增加对神经元有毒性。然而, 由于缺乏对患者的样本进行评价的方法, 因此无法研究在家族性/偶发性 PD 患者中是否改变了生理相关 LRRK2 基质的磷酸化。
蛋白质磷酸化通常是通过免疫或酶联免疫吸附试验 (ELISA), 使用抗体专门识别磷酸化状态的蛋白质或质谱分析。然而, 前者的策略有时不能应用, 因为在创建磷酸化特异抗体的困难。代谢标记的细胞与放射性磷酸盐是另一个选择, 以检查的生理水平磷酸化特定抗体是不容易获得。然而, 它需要大量的放射性材料, 因此涉及一些专用设备, 用于防护14。与这些免疫方法相比, 质谱分析更加灵敏, 在蛋白质磷酸化分析中得到了广泛的应用。然而, 样品准备是费时的, 并且昂贵的仪器是需要的分析。
酶家族的一个子集, 包括 Rab10 和 Rab8, 最近被报告为 LRRK2 的直接生理基质, 根据 large-scale phosphoproteomic 分析结果15。然后我们证明, Rab10 磷酸化是增加了小鼠胚胎成纤维细胞的平板的突变和在敲鼠标的肺部16。在这份报告中, 我们选择采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (sds 页) 为基础的方法, 其中 p 标记分子 co-polymerized 到 sds 页凝胶 (p 标签 sds 页), 以检测内源水平的 Rab10 磷酸化,因为对磷酸化 Rab10 特异性强的抗体仍然缺乏。我们没有检测到内源性 Rab8 的磷酸化, 由于目前可用的抗体对总 Rab8 的选择性较差。因此, 我们决定专注于 Rab10 磷酸化。LRRK2 对 Rab10 在 Thr73 位于高度保守的 "开关 II" 区域的中间。高保护的磷酸化点之间的蛋白质可能是一个原因, phosphospecific 抗体识别不同的蛋白质是很难作出的。
Rab8A 的磷酸化 LRRK2 抑制 Rabin8 的结合, 一种鸟嘌呤核苷酸交换因子 (GEF), 它通过与 GTP15交换绑定的 GDP 来激活 Rab8A。LRRK2 的磷酸化 Rab10 和 Rab8A 也抑制了国内生产总值-离解抑制剂 (GDIs) 的结合, 这对于通过从膜中提取国产总值束缚的蛋白从15中活化蛋白质是必不可少的。总的来说, 它是假设, LRRK2 的蛋白质磷酸化防止他们的活化, 虽然确切的分子机制和生理后果的磷酸化仍然不清楚。
P 标记 SDS-页是由木et al.在2006年发明的: 在这种方法中, 丙烯酰胺与 P 标记共价结合, 一个分子捕获高亲和力的磷酸盐, 共聚成 sds-页凝胶17。由于 sds 页凝胶中的 p 标记分子有选择地延缓磷酸化蛋白的电泳迁移, p 标记 sds 页可以将磷酸化蛋白与 non-phosphorylated 的蛋白质分离 (图 1)。如果 protein-of-interest 在多个残留物上磷酸化, 则会观察到相应的磷酸酯形态的阶梯。在 Rab10 的情况下, 我们只观察一个移位带, 表明 Rab10 是磷酸化只在 Thr73。P 标记 SDS 页的主要优势, 免疫与磷酸化特异抗体是, 磷酸化 Rab10 可以检测免疫与 non-phosphorylation 特异抗体 (即, 识别总 Rab10)在被转移的细胞膜之后, 通常是更加具体, 敏感, 并且可利用从商业或学术来源。使用 P 标记 SDS 页的另一个优点是, 可以获得对磷酸化的化学计量的近似估计, 这是不可能的免疫与磷酸化特异性抗体或代谢标记细胞与放射性磷酸盐.
除了使用廉价的 P 标记丙烯酰胺和一些小的修改与它相关, 目前的方法检测 Rab10 磷酸化的 LRRK2 遵循的一般协议免疫。因此, 它应该是直接和易于执行在任何实验室的免疫是一个通常的做法, 与任何类型的样品, 包括纯化蛋白, 细胞裂解, 和组织匀。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>homemade</td><td></td><td>150 mM NaCl, 8 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-12H&lt;sub&gt;2&lt;/sub&gt;O, 2.7 mM KCl, 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;in MilliQ water and sterilized by autoclaving</td></tr><tr><td>Sodium chloride</td><td>Nacalai Tesque</td><td>31320-34</td><td></td></tr><tr><td>Sodium Disodium Hydrogenphosphate 12-Water</td><td>Wako</td><td>196-02835</td><td></td></tr><tr><td>Potassium chloride</td><td>Wako</td><td>163-03545</td><td></td></tr><tr><td>Potassium Dihydrogen Phosphate</td><td>Wako</td><td>169-04245</td><td></td></tr><tr><td>2.5% Trypsin (10X)</td><td>Sigma-Aldrich</td><td>T4549</td><td>Dilute 10-fold with sterile DPBS for preparing working solution</td></tr><tr><td>Dulbecco&#039;s modified Eagle medium&lt;br/&gt; (DMEM)</td><td>Wako</td><td>044-29765</td><td></td></tr><tr><td>Fetal bovine serum</td><td>BioWest</td><td>S1560</td><td>Heat-inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Wako</td><td>168-23191</td><td></td></tr><tr><td>HEPES</td><td>Wako</td><td>342-01375</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Wako</td><td>198-13765</td><td></td></tr><tr><td>Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000)</td><td>PolySciences, Inc.</td><td>24765-1</td><td>Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH</td></tr><tr><td>Dimethyl sulfoxide</td><td>Wako</td><td>045-28335</td><td></td></tr><tr><td>Tris</td><td>STAR</td><td>RSP-THA500G</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako</td><td>080-01066</td><td></td></tr><tr><td>Polyoxyethylene(10) Octylphenyl Ether</td><td>Wako</td><td>160-24751</td><td>Equivalent to Triton X-100</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetraacetic acid (EGTA)</td><td>Wako</td><td>346-01312</td><td></td></tr><tr><td>Sodium orthovanadate(V)</td><td>Wako</td><td>198-09752</td><td></td></tr><tr><td>Sodium fluoride</td><td>Kanto Chemical</td><td>37174-20</td><td></td></tr><tr><td>&amp;beta;-Glycerophosphoric Acid Disodium Salt Pentahydrate</td><td>Nacalai Tesque</td><td>17103-82</td><td></td></tr><tr><td>Sodium pyrophosphate decahydrate</td><td>Kokusan Chemical</td><td>2113899</td><td></td></tr><tr><td>Microcystin-LR</td><td>Wako</td><td>136-12241</td><td></td></tr><tr><td>Sucrose</td><td>Wako</td><td>196-00015</td><td></td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td>Dissolve one tablet in 1 mL water, which can be stored at -20 &amp;deg;C for a month. Use it at 1:50 dilution for cell lysis</td></tr><tr><td>Pierce Coomassie (Bradford) Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23200</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Nacalai Tesque</td><td>31607-65</td><td></td></tr><tr><td>Glycerol</td><td>Wako</td><td>075-00616</td><td></td></tr><tr><td>Bromophenol blue</td><td>Wako</td><td>021-02911</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Kanto Chemical</td><td>25099-00</td><td></td></tr><tr><td>Ethanol</td><td>Wako</td><td>056-06967</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>136-01837</td><td></td></tr><tr><td>Phosphate-binding tag acrylamide</td><td>Wako</td><td>AAL-107</td><td>P-tag acrylamide</td></tr><tr><td>40% (w/v) acrylamide solution</td><td>Nacalai Tesque</td><td>06119-45</td><td>Acrylamide:Bis = 29:1</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Nacalai Tesque</td><td>33401-72</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Wako</td><td>016-08021</td><td>10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water</td></tr><tr><td>2-propanol</td><td>Wako</td><td>166-04831</td><td></td></tr><tr><td>Manganese chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M3634</td><td></td></tr><tr><td>Precision Plus Protein Prestained Standard</td><td>Bio-Rad</td><td>1610374, 1610373, 1610377</td><td>Molecular weight marker used in the protocol</td></tr><tr><td>WIDE-VIEW Prestained Protein Size Marker III</td><td>Wako</td><td>230-02461</td><td></td></tr><tr><td>Glycine</td><td>Nacalai Tesque</td><td>17109-64</td><td></td></tr><tr><td>Amersham Protran NC 0.45</td><td>GE Healthcare</td><td>10600007</td><td>Nitrocellulose membrane</td></tr><tr><td>Durapore Membrane Filter</td><td>EMD Millipore</td><td>GVHP00010</td><td>PVDF membrane</td></tr><tr><td>Filter Papers No.1</td><td>Advantec</td><td>00013600</td><td></td></tr><tr><td>Ponceau S</td><td>Nacalai Tesque</td><td>28322-72</td><td></td></tr><tr><td>Acetic acid</td><td>Wako</td><td>017-00251</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td>polyoxyethylenesorbitan monolaurate</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Wako</td><td>345-01865</td><td></td></tr><tr><td>Skim milk powder</td><td>Difco Laboratories</td><td>232100</td><td></td></tr><tr><td>Immunostar</td><td>Wako</td><td>291-55203</td><td>ECL solution (Normal sensitivity)</td></tr><tr><td>Immunostar LD</td><td>Wako</td><td>290-69904</td><td>ECL solution (High sensitivity)</td></tr><tr><td>CBB staining solution</td><td>homemade</td><td></td><td>1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water</td></tr><tr><td>CBB R-250</td><td>Wako</td><td>031-17922</td><td></td></tr><tr><td>CBB destaining solution</td><td>homemade</td><td></td><td>12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-HA antibody</td><td>Sigma-Aldrich</td><td>11583816001</td><td>Used at 0.2 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-Rab10 antibody</td><td>Cell Signaling Technology</td><td>#8127</td><td>Used at 1:1000 for immunoblotting.&lt;br/&gt; Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016.</td></tr><tr><td>anti-pSer935 antibody</td><td>Abcam</td><td>ab133450</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-LRRK2 antibody</td><td>Abcam</td><td>ab133518</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody</td><td>Sigma-Aldrich</td><td>T9026</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-GAPDH antibody</td><td>Santa-Cruz</td><td>sc-32233</td><td>Used at 0.02 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>515-035-003</td><td>Used at 0.16 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-035-003</td><td>Used at 0.16 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Inhibitors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GSK2578215A</td><td>MedChem Express</td><td>HY-13237</td><td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &amp;deg;C</td></tr><tr><td>MLi-2</td><td>Provided by Dr Dario Alessi (University of Dundee)</td><td></td><td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rab10/pcDNA5 FRT TO HA</td><td>Provided by Dr Dario Alessi&lt;br/&gt; (University of Dundee)</td><td></td><td>This plasmid expresses amino-terminally HA-tagged human Rab10.</td></tr><tr><td>LRRK2 WT/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Ito et al., Biochemistry, 46: 1380&amp;ndash;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2.</td></tr><tr><td>LRRK2 K1906M/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Ito et al., Biochemistry, 46: 1380&amp;ndash;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2.</td></tr><tr><td>LRRK2 N1437H/p3xFLAG-CMV-10</td><td></td><td></td><td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441C/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441G/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441H/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441S/p3xFLAG-CMV-10</td><td></td><td></td><td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 Y1699C/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 G2019S/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 I2020T/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific</td><td>Forma Series II 3110 Water-Jacketed</td><td></td></tr><tr><td>Auto Pipette</td><td>Drummond</td><td>Pipet-Aid PA-400</td><td></td></tr><tr><td>Micropipette P10</td><td>Nichiryo</td><td>00-NPX2-10</td><td>0.5&amp;ndash;10 &amp;mu;L</td></tr><tr><td>Micropipette P200</td><td>Nichiryo</td><td>00-NPX2-200</td><td>20&amp;ndash;200 &amp;mu;L</td></tr><tr><td>Micropipette P1000</td><td>Nichiryo</td><td>00-NPX2-1000</td><td>100&amp;ndash;1000 &amp;mu;L</td></tr><tr><td>Tips for micropipette P10</td><td>STAR</td><td>RST-481LCRST</td><td>Sterile</td></tr><tr><td>Tips for micropipette P200</td><td>FUKAEKASEI</td><td>1201-705YS</td><td>Sterile</td></tr><tr><td>Tips for micropipette P1000</td><td>STAR</td><td>RST-4810BRST</td><td>Sterile</td></tr><tr><td>5 mL disporsable pipette</td><td>Greiner</td><td>606180</td><td>Sterile</td></tr><tr><td>10 mL disporsable pipette</td><td>Greiner</td><td>607180</td><td>Sterile</td></tr><tr><td>25 mL disporsable pipette</td><td>Falcon</td><td>357535</td><td>Sterile</td></tr><tr><td>Hematocytometer</td><td>Sunlead Glass</td><td>A126</td><td>Improved Neubeuer</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CKX53</td><td></td></tr><tr><td>10 cm dishes</td><td>Falcon</td><td>353003</td><td>For tissue culture</td></tr><tr><td>6-well plates</td><td>AGC Techno Glass</td><td>3810-006</td><td>For tissue culture</td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>Vortex-Genie 2</td><td></td></tr><tr><td>Cell scrapers</td><td>Sumitomo Bakelite</td><td>MS-93100</td><td></td></tr><tr><td>1.5 mL tubes</td><td>STAR</td><td>RSV-MTT1.5</td><td></td></tr><tr><td>15 mL tubes</td><td>AGC Techno Glass</td><td>2323-015</td><td></td></tr><tr><td>50 mL tubes</td><td>AGC Techno Glass</td><td>2343-050</td><td></td></tr><tr><td>Centrifuges</td><td>TOMY</td><td>MX-307</td><td></td></tr><tr><td>96-well plates</td><td>Greiner</td><td>655061</td><td>Not for tissue culture</td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>SpectraMax M2e</td><td></td></tr><tr><td>SDS&amp;ndash;PAGE tanks</td><td>Nihon Eido</td><td>NA-1010</td><td></td></tr><tr><td>Transfer tanks</td><td>Nihon Eido</td><td>NA-1510B</td><td></td></tr><tr><td>Gel plates (notched)</td><td>Nihon Eido</td><td>NA-1000-1</td><td></td></tr><tr><td>Gel plates (plain)</td><td>Nihon Eido</td><td>NA-1000-2</td><td></td></tr><tr><td>Silicon spacers</td><td>Nihon Eido</td><td>NA-1000-16</td><td></td></tr><tr><td>17-well combs</td><td>Nihon Eido</td><td>Custom made</td><td></td></tr><tr><td>Binder clips</td><td>Nihon Eido</td><td>NA-1000-15</td><td></td></tr><tr><td>5 mL syringe</td><td>Terumo</td><td>SS-05SZ</td><td></td></tr><tr><td>21G</td><td>Terumo</td><td>NN-2138R</td><td></td></tr><tr><td>Power Station 1000 VC</td><td>ATTO</td><td>AE-8450</td><td>Power supply for SDS&amp;ndash;PAGE and transfer</td></tr><tr><td>Large weighing boats</td><td>Ina Optika</td><td>AS-DL</td><td></td></tr><tr><td>Plastic containers</td><td>AS ONE</td><td>PS CASE No.4</td><td>10 x 80 x 50 mm</td></tr><tr><td>Rocking shaker</td><td>Titech</td><td>NR-10</td><td></td></tr><tr><td>Styrene foam box</td><td>generic</td><td></td><td>The internal dimensions should fit one transfer tank (200 x 250 x 250 mm).</td></tr><tr><td>ImageQuant LAS-4000</td><td>GE Healthcare</td><td></td><td>An imager equipped with a cooled CCD camera for detection of ECL</td></tr></tbody></table>

rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag

富含亮氨酸重复激酶 2 (LRRK2) 的突变被证实与家族性帕金森病 (平板) 有关。由于 LRRK2 激酶活性的异常活化已经牵连到 PD 的发病机制中, 因此建立一种评价 LRRK2 激酶活性的方法是十分必要的。最近的研究表明, LRRK2 对酶家族的成员, 包括 Rab10, 在生理条件下。虽然在培养细胞中 LRRK2 内源性 Rab10 的磷酸化可以通过质谱法检测, 但由于目前可用的磷酸化特异抗体的敏感性差, 很难通过免疫检测Rab10在这里, 我们描述了一个简单的方法, 检测 Rab10 磷酸化的 LRRK2 基于免疫利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 结合磷酸盐结合标签 (P 标签), 这为N-(5-(2-aminoethylcarbamoyl) 吡啶-2-ylmetyl)-n,n ",n"-三 (吡啶-2-基-甲基)-13-diaminopropan-2-ol。本议定书不仅提供了利用 P 标记的方法的一个例子, 而且还能够评估突变以及抑制剂治疗/管理或任何其他因素如何改变 LRRK2 在细胞和组织中的下游信号.

过度表达系统: HA-Rab10 的磷酸化 3×FLAG-LRRK2 在 HEK293 细胞:
HEK293 细胞转染0.266 µg HA-Rab10 野生型和1.066 µg 3×FLAG-LRRK2 (野生型, 激酶-不活泼突变体 (K1906M), 或平板变种)。Rab10 磷酸化检查的 P 标记 SDS 页后, 免疫使用抗 HA 抗体 (图 2)。10µg 蛋白质是运行在10% 凝胶 (80 x 100 x 1 毫米) 包含50µM P 标记丙烯酰胺和100µM MnCl<...

Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

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

本研究介绍了一种简单的方法检测内源性水平的 Rab10 磷酸化富亮氨酸重复激酶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 ...

rab10-phosphorylation-detection-lrrk2-activity-using-sds-page-with

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

14.9K 次观看.  The University of Tokyo. 本研究介绍了一种简单的方法检测内源性水平的 Rab10 磷酸化富亮氨酸重复激酶2。

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

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

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

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 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

生物学

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Source: Bailey, E. M. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> J. Vis. Exp. (2022)
This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

用于蛋白质磷酸化分析的单分子沉降测定:一种定量细胞裂解物中蛋白质磷酸化的高通量技术

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)
This video ...