我们感谢张庆信教授对缺氧工作站的使用。这项工作得到以下方面的支持: 新加坡教育部、教育部 1T1-02/04 和 MOE2015-T2-2-087 (Y.A.)、李港医学院、南洋理工大学开办补助金 M4230003 (P.O.B.)、瑞典研究理事会、家庭苓基金会, 诺德基金会, Stichting af Jochnick 基金会, 瑞典糖尿病协会, 氧化钪保险公司, 糖尿病研究和健康基金会, 泊位冯 Kantzow 的基础,战略研究计划在糖尿病卡罗林斯卡 Institutet, 紧急救济 ERC-2013-AdG 338936 Betalmage, 和克努特和爱丽丝拉乌尔·沃伦贝格基金会。

本协议部分采用人类胚胎肾脏 293A (HEK293A) 细胞, 遵循新加坡南洋理工大学人类研究伦理学委员会的指导方针。
1. HEK293A 细胞缺氧的诱导
<ol><li>准备四10厘米的菜肴和种子 3–5 x 106 HEK293A 细胞每盘在10毫升 Dulbecco 的改良鹰的培养基 (DMEM, 4.5 克/升葡萄糖) 辅以10% 胎牛血清 (血清), 2 毫米 l-谷氨酰胺, 110 毫克/升丙酮钠, 100 U/毫升青霉素和100毫克/链霉素。培养细胞在37°c, 5% CO2孵化器。 注: 细胞播种应在生物安全柜 (BSC) 进行。要放置在平衡计分卡上的所有材料的表面应用70% 乙醇擦拭。</li>
	<li>24小时播种后, 当细胞达到80–90% 融合时, 将两个盘子放入孵化 glovebox 中的缺氧 subchamber (见材料表) 与 1% O2和 5% CO2在37°c, 并保持其他两个盘子在 normoxia (21% O2, 5% co2在37°c) 为 4 h. 利用一组常氧和缺氧细胞对免疫沉淀的低氧治疗进行评价, 并利用另一组细胞进行联合实验。 注: 氧水平可以设定在 0–5%, 缺氧治疗的持续时间可以根据细胞类型和研究目标而变化。</li>
</ol>2. 全细胞和核萃取
注: 有关此协议中使用的缓冲区的信息, 请参见表 1 。
<ol><li>
收获 normoxia 控制细胞。
	<ol>
<li>用10毫升 DPBS (PH 7.0–7.2) 用10毫升的吸管去除培养基, 用吸入法冲洗细胞。 注意: 避免用吸管接触细胞单层。在洗涤过程中, 轻轻地将 DPBS 在细胞培养板的壁上, 以避免细胞流失。</li>
		<li>吸管5毫升冰冷 DPBS 进入板和刮的细胞表面上的冰冷 PBS 与细胞刮刀。</li>
		<li>将细胞悬浮液转移到15毫升锥形管中, 并保持在冰上。</li>
	</ol>
</li>
	<li>
在缺氧条件下收获细胞培养。
	<ol>
<li>预平衡的 DPBS 到缺氧条件通过放置一个发现100 毫升实验玻璃试剂瓶填充 DPBS 在缺氧 subchamber (1% O2和 5% CO 2 在 37 °c), 提前24小时。</li>
		<li>大约 1 h 在收获缺氧治疗的细胞之前, 放置一个冰盒包含冰入 glovebox 的处理室, 已经平衡到 1% O2和 5% CO2。将含有预平衡缺氧 DPBS 的瓶子从缺氧 subchamber 转移到处理室并放在冰上。</li>
		<li>低氧治疗后4小时, 将细胞从缺氧 subchamber 转移到平衡前 1% O2和 5% CO2的处理室。</li>
		<li>通过吸入去除培养基, 用10毫升冰冷预平衡缺氧 DPBS 和10毫升吸管冲洗细胞一次。</li>
		<li>添加5毫升冰冷预平衡 DPBS 与5毫升吸管, 并通过刮一个细胞刮刀去除细胞。</li>
		<li>倾斜细胞培养板和收集分离细胞使用10毫升吸管。将 DPBS 中的细胞悬浮液转移到15毫升锥形管中, 并保持在冰上。</li>
		<li>打开 glovebox 的处理和缓冲室之间的门, 两者都已预平衡到 1% O2 和 5% CO2。将含有缺氧处理细胞的冰上的15毫升锥形管转移到缓冲室。打开缓冲室的门, 完全从 glovebox 中取出细胞。</li>
	</ol>
</li>
	<li>常氧细胞从2.1 和缺氧细胞从2.2 通过离心在 1000 x g为5分钟在4°c。</li>
	<li>
准备整个细胞提取物。
	<ol>
<li>并用重悬500µL 中的细胞颗粒, 包括50毫米 Trisaminomethane 盐酸盐 (三盐酸) pH 8.0、150毫米氯化钠 (NaCl)、1% tergitol 型 NP-40 (NP-40)、0.5% 钠脱氧和0.1% 月桂酸钠 (SDS), 辅以1x 蛋白酶抑制剂鸡尾酒 (见材料表) 通过吹打细胞颗粒上下几次。</li>
		<li>将细胞裂解物转移到新的1.5 毫升离心管中, 并在冰上保持30分钟, 偶尔涡流。</li>
		<li>离心细胞裂解物在 1.3万 x g 10 分钟在4°c。</li>
		<li>收集上清, 整除50µL 的上清到1.5 毫升离心管, 并存储在-80 摄氏度。</li>
	</ol>
</li>
	<li>
用核萃取试剂盒准备核提取物 (见材料表)。
	<ol>
<li>用1x 蛋白酶抑制剂鸡尾酒和0.1 米 dithiothreitol (并用重悬) 在500µL 的裂解缓冲液中轻轻地将细胞颗粒向上和向下吹打几次。</li>
		<li>在十年代以最大速度添加25µL 的洗涤剂溶液 NP 到细胞悬浮和漩涡。</li>
		<li>离心机在 1万 x g 5 分钟在4摄氏度。</li>
		<li>收集上清 (细胞质提取物), 整除50µL 的上清到1.5 毫升离心管, 并贮存在-80 摄氏度。</li>
		<li>并用重悬500µL 裂解缓冲物中含有细胞核的球团, 辅以1x 蛋白酶抑制剂鸡尾酒和0.1 米的涡流, 最大速度为5秒。</li>
		<li>离心机在 1万 x g 5 分钟在4°c 和保存核球团。</li>
		<li>并用重悬50µL 萃取缓冲液中的核球团 NX1 辅以1x 蛋白酶抑制剂鸡尾酒, 多次吹打颗粒。</li>
		<li>在冰上孵化30分钟, 涡流为十年代每5分钟在最大速度。</li>
		<li>离心机在 1.2万 x g 10 分钟在4摄氏度。</li>
	</ol>
</li>
	<li>
淡化核萃取物。
	<ol>
<li>从步骤2.5.9 收集上清液, 并转入微型透析装置, 最大容积为每单位100µL。</li>
		<li>将微型透析装置盖上, 放在浮选装置中。</li>
		<li>将浮选装置放在装有500毫升预冷透析缓冲液的烧杯中 (20mM 三盐酸 pH 7.4, 20% 甘油, 100mM 氯化钾 (氯化钾), 0.2 毫米乙二胺二乙酸 (EDTA), 0.2 毫米 phenylmethylsulfonyl 氟 (PMSF) 和0.5 毫米)并孵育30分钟, 在4摄氏度与温和搅拌。 警告: PMSF 是危险的。避免直接接触皮肤或吸入。</li>
		<li>收集的样品从角落的迷你透析设备和转移到新的1.5 毫升离心管。</li>
		<li>离心机在 1.2万 x g 10 分钟在4°c, 整除25µL 每上清入1.5 毫升离心管, 并且存放在-80 °c。</li>
	</ol>
</li>
</ol>3. HIF-1α蛋白表达和亚细胞定位评价低氧治疗的价值
<ol><li>根据制造商的说明18, 测定二辛可宁酸 (微板块) 蛋白检测试剂盒的全细胞或核/细胞质提取物的蛋白质浓度。</li>
	<li>稀释细胞裂解物在 1x Laemmli 样品缓冲包含 5% 2-巯基乙醇并且煮沸在95°c 为5分钟。 注意: 不要接触加热块的表面, 因为它可能会引起烧伤。</li>
	<li>
用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分离蛋白质。
	<ol>
<li>将等量的蛋白质 (25 µg) 加载到 SDS 页预制梯度凝胶 (4–20%) 的每个井中, 以及分子量标记的3µL。</li>
		<li>运行的凝胶在运行缓冲区中含有2.5 毫米三, 19.2 毫米甘氨酸, 0.01% SDS, PH8.3 30 分钟 200 v。</li>
	</ol>
</li>
	<li>
将蛋白质从凝胶转移到硝化膜。
	<ol>
<li>将转移夹层 (过滤纸-凝胶膜过滤纸) 与阳极边上的凝胶和卡带负极端的膜组装起来。</li>
		<li>将卡带放入装有2.5 毫米 trisaminomethane (三)、19.2 mm 甘氨酸和20% 甲醇的转移缓冲器的转储罐中。 注意: 甲醇是易燃的, 应储存在易燃液体贮存柜内。</li>
		<li>在冷室的100伏处进行1小时的转移。</li>
	</ol>
</li>
	<li>在10毫升的阻挡缓冲器中, 用50毫米三氯 (pH 7.6), 150 毫米氯化钠, 0.1 吐温20和5% 个非脂牛奶, 在室温下在振动筛上阻挡印迹。</li>
	<li>用 anti-HIF-1α抗体 (1/500 稀释) 在4摄氏度的同一阻塞缓冲液中孵化出印迹。</li>
	<li>每次用50毫升的3次清洗污点5分钟。</li>
	<li>用辣根过氧化物酶 (HRP)-共轭二级抗体 (1/1000 稀释) 在室温下用10毫升含5% 非脂肪干牛奶的1小时孵化出印迹。</li>
	<li>每次用50毫升的3次清洗污点5分钟。</li>
	<li>将500µL 的增强 chemilumescent (ECL) 试剂 a 和 B 混合在1.5 毫升离心管和涡流中。</li>
	<li>在室温下将 ECL 基底涂抹在印迹上, 孵育1分钟。</li>
	<li>
利用电荷耦合器件 (CCD) 摄像成像系统捕获化学发光信号。
	<ol>
<li>通过接触膜的边缘与纸巾, 将膜放在板料保护器中, 排出多余的 ECL 基体。</li>
		<li>将膜放在 CCD 摄像成像系统的样品托盘上。</li>
		<li>启动图像处理软件 (参见材料表), 并使用以下设置捕获图像: 文件→新协议→单通道→协议设置→凝胶成像 (应用: 化学;成像区域: 生物 Rad 准备凝胶;成像曝光: 软件将自动优化的曝光时间为强频带) →运行协议 注意: 可以手动设置曝光时间, 以实现最佳图像。</li>
	</ol>
</li>
</ol>4. Immunoprecipitated 蛋白的免疫沉淀和检测
<ol><li>在1.5 毫升离心管中, 将50µL 的蛋白 A/G 琼脂糖珠在500µL 的 tb 缓冲器中洗净, 用离心法在 3000 x G上以2°c 的方法将珠子颗粒化。</li>
	<li>在100µL 的 tb 缓冲器中, 丢弃上清和并用重悬珠子。</li>
	<li>添加2µL 的小鼠单克隆抗 ARNT 抗体 (1.4 毫克/毫升) 或小鼠免疫球蛋白 G (IgG) (1.4 毫克/毫升), 通过重组0.7 毫克鼠 IgG 在500µL tb 缓冲。</li>
	<li>放置1.5 毫升的离心管, 其中包含珠在管转子和孵化2小时在 10 rpm 在冷室。 注意: 轻轻地处理管子, 并保持悬浮在管子底部的珠子。</li>
	<li>以 3000 x g为2分钟, 在4摄氏度处离心成珠, 然后丢弃上清液。</li>
	<li>稀释200µg 在步骤2.6.5 中获得的核蛋白裂解物, 其800µL 的 IP 缓冲器由50毫米三盐酸 (pH 7.4)、180毫米氯化钠、20% 甘油、0.2% NP-40 和1x 蛋白酶抑制剂鸡尾酒组成。在4摄氏度, 在步骤4.5 中获得的抗体耦合珠孵化出裂解液。</li>
	<li>以 3000 x g为2分钟, 在4摄氏度时, 将珠子颗粒离心, 丢弃上清液, 用1毫升冰冷 tb (含 0.2% NP-40) 将珠子洗净3次。</li>
	<li>将珠子煮沸50µL Laemmli 样品缓冲器95°c 5 分钟。</li>
	<li>离心 1万 x g 5 分钟的珠子在4摄氏度, 收集上清, 并丢弃珠。 注: 上清液可储存在4摄氏度, 短期或-20 摄氏度长期。</li>
	<li>从 immunoprecipitated 蛋白配合物中检测出 HIF-1α的存在, 如上文步骤3.3 所述。 注: 此步骤不需要蛋白质量化。将每个样品上清的全部容积 (50 µL) 装入 SDS 页预制梯度凝胶 (4–20%) 的每个井中。</li>
</ol>

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Y., Powis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passing+the+baton:+The+HIF+switch.">Passing the baton: The HIF switch.</a> Trends Biochem Sci. 37 (9), 364-372 (2012).</li><li>Dumetz, A. C., Snellinger-O'brien, A. M., Kaler, E. W., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+protein+protein+interactions+in+salt+solutions+and+implications+for+protein+crystallization.">Patterns of protein protein interactions in salt solutions and implications for protein crystallization.</a> Protein Sci. 16 (9), 1867-1877 (2007).</li><li>Graven, K. K., Troxler, R. F., Kornfeld, H., Panchenko, M. V., Farber, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endothelial+cell+glyceraldehyde-3-phosphate+dehydrogenase+expression+by+hypoxia.">Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.</a> J Biol Chem. 269 (39), 24446-24453 (1994).</li><li>Caradec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desperate+house+genes':+The+dramatic+example+of+hypoxia.">Desperate house genes': The dramatic example of hypoxia.</a> Br J Cancer. 102 (6), 1037-1043 (2010).</li></ol>

作者声明没有利益冲突。

HIF-1 复合体是细胞氧稳态的主调节器, 它调节了多种细胞适应缺氧反应的基因。新的 HIF-1 相互作用蛋白的鉴定对于了解缺氧信号转导具有重要意义。免疫沉淀实验通常用于 PPIs 研究, 描绘细胞信号转导通路。然而, 蛋白质过度表达仍然被广泛使用, 这可能导致实验性工件。此外, HIF-1α是一种高度不稳定的蛋白质, 它在重氧11期间迅速降解。此外, 缺氧会触发大多数哺乳动物细胞系中 HIF-1 成分的核易位。因此, 必须对常规的免疫沉淀协议进行优化, 以确定缺氧治疗后生理相关的 HIF-1 相互作用蛋白。在这里, 我们描述了一个改进的免疫沉淀协议, 用来证明 HIF-1α和 ARNT 在缺氧之间的相互作用。
为避免 HIF-1α在再氧过程中的降解, 在 glovebox 的培养室中, 缺氧细胞在平衡缺氧条件下被收获。DPBS 洗涤缓冲器在使用前也平衡缺氧条件。如果没有缺氧工作站可供加工, 则可以用预平衡缺氧 DPBS 清洗缺氧细胞, 然后在冰上迅速收获。考虑到缺氧可触发 HIF-1 成分的核易位, 而蛋白过度表达可能诱发 artefactual 结果, 本协议使用内源核蛋白。其他20人也报告了在共同免疫沉淀议定书中使用核萃取物的情况。它代表了研究核蛋白之间相互作用的技术优势, 因为它最大限度地减少了核蛋白被全细胞蛋白稀释的几率。此外, 使用核萃取物可能有助于减少非特异性的相互作用, 特别是从富含大量无关的细胞质蛋白, 并通过最大限度地减少接触到核蛋白的稳定性细胞质中存在蛋白酶。使用内源核萃取物代替全细胞提取物对免疫沉淀显示了显著的技术改进, 因为我们只能检测 HIF-1α和 GABPα使用内源核萃取物之间的相互作用, 但不使用整体细胞提取物17。
在描述的协议中, 可以进一步优化几个条件以提高结果。以1% 氧为4小时, 诱导 HEK293A 细胞缺氧。但是氧水平和缺氧治疗的持续时间可以不同的细胞类型, 并根据研究的目的进行调整。例如, HIF-1α和 HIF-2α可以在细胞类型的特定方式下对缺氧进行差异调节, 表明它们在不同生物学环境中的独特作用。结果表明, 在神经母细胞瘤中, HIF-1α在短时间的剧烈缺氧 (1% o2) 时最活跃, 而 HIF-2α在轻度缺氧 (5% o2) 下活跃, 在慢性缺氧治疗后变得更加活跃 (48–72缺氧暴露)21。0–5% o2水平通常用于体外缺氧治疗, 其中 1–5% o2, 0.1–1%2和 0–0.1% o2经常被定义为轻度缺氧, 缺氧和缺氧, 分别为22。盐浓度是另一个需要优化的参数, 特别是因为它在 PPIs23中对离子相互作用起着关键作用。在这项研究中使用核萃取物, 通常用含有高浓度盐的缓冲液提取核蛋白。因此, 在共同免疫沉淀实验之前, 核萃取物可能必须淡化。在这里, 我们用含有100毫米氯化钾的透析缓冲器淡化核萃取物, 并将透析提取物与含有180毫米氯化钠的 IP 缓冲器混合, 以达到接近150毫米的最终盐浓度, 这与生理细胞内 PPIs 条件。最后的盐浓度可以根据 PPIs 的性质进行修改。在本协议中, GAPDH 被用作整个细胞提取物和细胞质组分的加载控制。我们没有观察到任何明显的缺氧诱导调节作用的 GAPDH 表达 HEK293A 细胞。然而, GAPDH 以前被证明是上调的缺氧在某些细胞类型24。有了这一点, 你可能需要使用替代适当的加载控制时, 必要的25。另外, 我们观察到在当前协议中使用的珠子或抗体所产生的大量背景信号。为了减少背景, 你可以延长洗涤步骤 (10–15分钟) 或执行超过3洗涤 (5–10时间)。另外, 洗涤缓冲液的离子强度也可以通过在洗涤过程中滴定盐浓度从150到500毫米来增加。裂解物也可以预先清除通过孵化与蛋白 A/G 琼脂糖珠1小时在4°c 与旋转。
本议定书仅限于核萃取物, 因此可能不适用于研究其他细胞室, 如线粒体和内质网中的 PPIs。类似于其他常规的免疫沉淀协议, 此方法不能用于实时研究 PPIs, 也不能用于确定 PPIs 是直接的还是间接的。
总之, 我们提供了一个改进的共同免疫沉淀协议, 以确定新的生理相关的 HIF-1 相互作用的蛋白质。该协议也适用于研究在缺氧条件下转录因子与转录联合调节剂之间的相互作用。虽然该协议是专门为在缺氧条件下进行的免疫沉淀实验而设计的, 但所描述的 normoxia 控制单元协议的一部分也可用于研究 normoxia 条件下的核 PPIs。

缺氧发生时, 没有足够的氧气供应给细胞和组织的身体。它在各种生理和病理过程中起着关键作用, 如干细胞分化、炎症和癌症1、2。缺氧诱导因子 (HIFs) 功能, heterodimers 由氧调节α亚基和组成性表达β亚基也称为 ARNT3。迄今已确定了 HIF α亚基 (HIF-1α、HIF-2α和 HIF-3α) 和三个 HIF β亚基 (ARNT/HIF-1β、ARNT2 和 ARNT3) 的三种异构体。HIF-1α和 ARNT 是无所不在表达的, 而 HIF-2α、HIF-3α、ARNT2 和 ARNT3 有更限制性的表达模式4。HIF-1 蛋白复合物是缺氧反应的关键调节因子。在缺氧条件下, HIF-1α变稳定, translocates 至细胞核, dimerizes ARNT5。随后, 这一复杂的结合到特定的核苷酸称为缺氧反应元素 (HREs), 并调节所涉及的目标基因的表达, 包括血管生成, 红细胞和糖酵解6。除了这种 "规范" 的反应, 缺氧信号通路也被称为串扰多细胞反应信号通路, 如缺口和核因子-卡伯 B (NF-nf-κb)7,8,9。
新的 HIF-1 相互作用蛋白的鉴定对于更好地理解缺氧信号通路是很重要的。与 ARNT, 这是不敏感的氧气水平和组成性表达, HIF-1α蛋白质水平是严格调节细胞氧水平。在 normoxia (21% 氧), HIF-1α蛋白质迅速退化10,11。HIF-1α在 normoxia 的短半衰期为细胞提取物中蛋白质的检测以及 HIF-1α-interacting 蛋白的鉴定提供了具体的技术挑战。此外, 一些转录因子, 包括那些 HIF-1 复合体植物常常将进入细胞核的缺氧条件下12,13,14。目前用于 PPI 研究的大多数方法都是使用非生理的蛋白质过度表达来进行的。这种蛋白过度表达通过多种机制 (包括资源超载、化学计量失衡、混杂相互作用和通路调制15、16) 引起了不同的细胞缺陷。在 PPI 研究方面, 蛋白质过度表达可能导致假阳性, 甚至是假阴性, 结果取决于蛋白质的性质和功能的抗原蛋白。因此, 目前的 PPI 研究方法必须修改, 以揭示在缺氧条件下的生理相关 PPIs。我们以前已经证明了 HIF-1 和 Ets 家族转录因子遗传结合蛋白 (GABP) 在缺氧 P19 细胞中的相互作用, 这有助于Hes1启动子对缺氧17的反应。在这里, 我们描述了一个共同免疫沉淀协议, 研究 PPIs 之间的内源性核蛋白在缺氧条件下。HIF-1α与 ARNT 的相互作用表现为概念的证明。该协议适用于证明转录因子与转录联合调节器在缺氧条件下的相互作用, 包括但不限于 HIF-1 相互作用蛋白的鉴定。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.0 M Tris-HCl Buffer, pH 7.4&amp;nbsp;</td><td>1st BASE</td><td>1415</td><td></td></tr><tr><td>Protein A/G Sepharose beads</td><td>Abcam</td><td>ab193262</td><td></td></tr><tr><td>Natural Mouse IgG protein</td><td>Abcam</td><td>ab198772</td><td></td></tr><tr><td>EDTA</td><td>Bio-Rad</td><td>1610729</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610737</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710</td><td></td></tr><tr><td>Nitrocellulose Membrane &amp;nbsp;&amp;nbsp;</td><td>Bio-Rad</td><td>1620112</td><td></td></tr><tr><td>Blotting-Grade Blocker</td><td>Bio-Rad</td><td>1706404</td><td>Non-fat dry milk for western blotting applications</td></tr><tr><td>10x Tris Buffered Saline (TBS)</td><td>Bio-Rad</td><td>1706435</td><td></td></tr><tr><td>10% Tween 20</td><td>Bio-Rad</td><td>1610781</td><td></td></tr><tr><td>10x Tris/Glycine/SDS</td><td>Bio-Rad</td><td>1610732</td><td></td></tr><tr><td>10x Tris/Glycine Buffer&amp;nbsp;</td><td>Bio-Rad</td><td>1610771</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standards</td><td>Bio-Rad</td><td>1610374</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody&amp;nbsp;</td><td>Cell Signaling</td><td>7076</td><td></td></tr><tr><td>SignalFire ECL Reagent</td><td>Cell Signaling</td><td>6883</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Merck Millipore</td><td>52332</td><td></td></tr><tr><td>ARNT/HIF-1 beta Antibody&amp;nbsp;</td><td>Novus Biologicals</td><td>NB100-124&amp;nbsp;</td><td>Concentration: 1.4 mg/mL</td></tr><tr><td>HIF-1 alpha Antibody</td><td>Novus Biologicals</td><td>NB100-479</td><td>Concentration: 1.0 mg/mL</td></tr><tr><td>YY1 Antibody</td><td>Novus Biologicals</td><td>NBP1-46218</td><td>Concentration: 0.2 mg/mL</td></tr><tr><td>Qproteome Nuclear Protein Kit</td><td>Qiagen</td><td>37582</td><td>Lysis buffer NL and Extraction Buffer NX1 are provied in the kit</td></tr><tr><td>GAPDH Antibody</td><td>Santa Cruz</td><td>sc-47724</td><td>Concentration: 0.2 mg/mL</td></tr><tr><td>Glycerol (&amp;ge;99%)</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma</td><td>71376</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>127087-87-0</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM, 4.5 g/L glucose)</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher Scientific</td><td>R0861</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>HEK293A cell line</td><td>Thermo Fisher Scientific</td><td>R70507</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>88660</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>QSP gel loading tip&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>QSP#010-R204-Q-PK</td><td>1-200 uL</td></tr><tr><td>&lt;strong&gt;Equipment/Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thick Blot Filter Paper, Precut, 7.5 x 10 cm</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot&amp;nbsp;Module and PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>1658034</td><td></td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Precast Protein Gels</td><td>Bio-Rad</td><td>4561083</td><td></td></tr><tr><td>ChemiDoc XRS+ System</td><td>Bio-Rad</td><td>1708265</td><td></td></tr><tr><td>I-Glove</td><td>BioSpherix</td><td>I-Glove</td><td></td></tr><tr><td>Synergy HTX Multi-Mode Microplate Reader&amp;nbsp;</td><td>BioTek</td><td>BTS1LFTA</td><td></td></tr><tr><td>Costar 5mL Stripette Serological Pipets</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>Costar 10mL Stripette Serological Pipets</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Costar 25mL Stripette Serological Pipets</td><td>Corning</td><td>4251</td><td></td></tr><tr><td>Corning 96-Well Clear Bottom Black Polystyrene Microplates</td><td>Corning</td><td>3631</td><td></td></tr><tr><td>15mL High Clarity PP conical Centrifuge Tubes</td><td>Corning</td><td>352095</td><td></td></tr><tr><td>Small Cell Scraper</td><td>Corning</td><td>3010</td><td></td></tr><tr><td>Gilson&amp;nbsp;Pipetman L 4-pipettes kit&amp;nbsp;</td><td>Gilson</td><td>F167370</td><td>P2, P20, P200, P1000 and accessories</td></tr><tr><td>1.5mL Polypropylene Microcentrifuge Tubes</td><td>Greiner Bio-One&amp;nbsp;</td><td>616201</td><td></td></tr><tr><td>PIPETBOY acu 2 Pipettor</td><td>INTEGRA Biosciences</td><td>155 000&amp;nbsp;</td><td></td></tr><tr><td>Justrite Flammable Liquid Storage Cabinets</td><td>Justrite Manufacturing Co.</td><td>896000</td><td></td></tr><tr><td>Vortex mixer</td><td>Labnet</td><td>S0200</td><td></td></tr><tr><td>CO2 incubator</td><td>NuAire</td><td>NU-5820</td><td></td></tr><tr><td>Orbital shakers</td><td>Stuart</td><td>SSL1</td><td></td></tr><tr><td>Tube rotator SB3</td><td>Stuart</td><td>SB3</td><td></td></tr><tr><td>MicroCL 21R Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75002470</td><td></td></tr><tr><td>Sorvall ST 16 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75004240</td><td></td></tr><tr><td>Tissue Culture Dishes (100 mm)</td><td>Thermo Fisher Scientific</td><td>150350</td><td></td></tr><tr><td>Slide-A-Lyzer MINI Dialysis Device</td><td>Thermo Fisher Scientific</td><td>69580</td><td>10K MWCO, 0.1 mL</td></tr><tr><td>Float Buoys for 0.1mL Slide-A-Lyzer MINI Dialysis Devices</td><td>Thermo Fisher Scientific</td><td>69588</td><td></td></tr><tr><td>LSE Digital Dry Bath Heaters</td><td>Thermo Fisher Scientific</td><td>1168H25</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;1300 Series A2 Class II, Type A2 Bio Safety Cabinets</td><td>Thermo Fisher Scientific</td><td>13-261-308</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Lab Software</td><td>Bio-Rad</td><td>1709691</td><td></td></tr></tbody></table>

co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

低氧水平 (缺氧) 触发多种适应反应与缺氧诱导因子 1 (HIF-1) 复合体作为主调节器。HIF-1 由 heterodimeric 氧调节α亚基 (HIF-1α) 和组成性表达的β亚基 (HIF-1β) 也称为芳烃受体核转运体 (ARNT), 调节包括血管生成在内的不同过程中所涉及的基因、红细胞和糖酵解。HIF-1 相互作用蛋白的鉴定是了解缺氧信号通路的关键。除了调节 HIF-1α稳定性外, 缺氧还触发了许多转录因子的核易位, 包括 HIF-1α和 ARNT。值得注意的是, 目前用于研究这种蛋白质-蛋白质相互作用 (PPIs) 的方法大多是基于那些通过蛋白质过度表达人为地增加蛋白质水平的系统。蛋白质过度表达往往导致非生理结果的时间和空间文物。在这里, 我们描述了在低氧治疗后使用内源核蛋白的改进的免疫沉淀协议, 并作为概念的证明, 以显示 HIF-1α和 ARNT 之间的相互作用。在本议定书中, 缺氧细胞是在缺氧条件下收获的, Dulbecco 的磷酸盐缓冲盐水 (DPBS) 洗涤缓冲液也在使用前平衡缺氧条件, 以减轻蛋白质降解或蛋白质复合体复氧过程中的离解。此外, 核分数随后提取, 以集中和稳定内源性核蛋白, 并避免可能的假结果往往看到在蛋白质过度表达。该协议可用于证明转录因子与转录联合调节器在缺氧条件下的内源和本机相互作用。

为了评估细胞对缺氧的反应, 研究了缺氧治疗后 HIF-1 复合物的表达水平和亚单位定位。HEK293A 细胞在缺氧条件下培养4小时或保持在 normoxia 作为控制。HIF-1α和 ARNT 蛋白水平检查在整个细胞或核/细胞质提取物的西方印迹。按照预期, 缺氧上调总 HIF-1α水平, 而 ARNT 总细胞裂解物水平没有显著改变 (图 1A)。此外, 缺氧导致 HEK293A 细胞中 HIF-1α和 ARNT 的核积累 (图 1B), 这与以前的报告一致, 尽管在一些经测试的细胞系中未发现 ARNT 的细胞质表达.
接下来, 我们展示了 HIF-1α和 ARNT 在缺氧治疗后的相互作用。核萃取物是从暴露于常氧或缺氧条件下的 HEK293A 细胞中制备的, 用于 4 h. 用 HEK293A 细胞核萃取物进行联合免疫沉淀实验。如图 2所示, HIF-1α与低氧 HEK293A 细胞核萃取物 immunoprecipitated 一起 ARNT。
结合起来, 所描述的协议可以成功地用于诱导细胞缺氧反应, 并进一步确定内在表达 HIF-1α/ARNT 复合体在细胞核内的生理蛋白结合。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57836/57836fig1.jpg"> 图 1:低氧对 HIF-1 组分蛋白表达和亚细胞定位的调节.(A) 对 HEK293A 细胞中总 HIF-1α或 ARNT 表达的应用免疫印迹分析。细胞暴露于缺氧4小时 (h) 或保持在 normoxia (N)。以 anti-HIF-1α、抗 ARNT 和抗甘油 3-磷酸脱氢酶 (GAPDH) 抗体为免疫印迹法, 用 SDS 分页分离出全细胞提取物中的蛋白质。GAPDH 被用作装载控制。(B) 应用免疫印迹分析 HEK293A 细胞亚组分 HIF-1α和 ARNT 表达。HEK293A 细胞在缺氧条件下培养4小时 (h) 或保存在 normoxia (N)。采用 anti-HIF-1α免疫印迹法、抗 ARNT、抗阴、阳 1 (YY1) 和抗 GAPDH 抗体, 对25µg 核或细胞质提取物中蛋白质进行了分析。YY1 和 GAPDH 分别用作核和细胞质馏分的加载控制。<a href="/files/ftp_upload/57836/57836fig1large.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/57836/57836fig2.jpg"> 图 2:HIF-1α在缺氧条件下与 ARNT 相互作用.HEK293A 细胞暴露于缺氧4小时或保持在 normoxia 作为控制。制备了 HEK293A 细胞的核萃取物, 并用抗 ARNT 抗体进行了 immunoprecipitations。采用 anti-HIF-1α、抗 ARNT、anti-YY1 抗体免疫印迹法, 对核萃取物 (输入) 和 immunoprecipitated 蛋白进行了分析。YY1 被用作输入的加载控制, 而 IgG 作为免疫沉淀实验的负控制。<a href="/files/ftp_upload/57836/57836fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>解决 方案</td>
			<td>组件</td>
			<td>评论</td>
		</tr>
<tr>
<td>透析缓冲液</td>
			<td>20毫米三盐酸 (pH 7.4), 20% 甘油, 100 毫米氯化钾, 0.2 毫米 EDTA, 0.2 毫米 PMSF 和0.5 毫米</td>
			<td>在使用前立即添加 PMSF 和德勤。</td>
		</tr>
<tr>
<td>运行缓冲区</td>
			<td>2.5 毫米三盐酸 (PH 8.3), 19.2 毫米甘氨酸和 0.01% SDS</td>
			<td>混合100毫升10x 三/甘氨酸/SDS 缓冲器与900毫升 ddH2O。</td>
		</tr>
<tr>
<td>传输缓冲区</td>
			<td>2.5 毫米三盐酸 (PH 8.3), 19.2 毫米甘氨酸和20% 甲醇</td>
			<td>混合100毫升10x 三/甘氨酸缓冲器与200毫升甲醇和700毫升 ddH2O。</td>
		</tr>
<tr>
<td>tb 缓冲区</td>
			<td>50毫米三氯 (pH 7.6) 和150毫米氯化钠</td>
			<td>混合100毫升 10x tb 缓冲器与900毫升 ddH2O。</td>
		</tr>
<tr>
<td>缓冲器</td>
			<td>50毫米三氯 (pH 7.6), 150 毫米氯化钠和0.1% 吐温20</td>
			<td>混合100毫升 10x tb 缓冲器与900毫升 ddH2O 和1毫升的吐温20。</td>
		</tr>
<tr>
<td>阻塞缓冲区</td>
			<td>50毫米三氯 (pH 7.6), 150 毫米氯化钠, 0.1% 吐温20和5% 非脂牛奶</td>
			<td>溶解5克的印迹级阻滞剂在100毫升的 tb 吨缓冲。</td>
		</tr>
<tr>
<td>IP 缓冲区</td>
			<td>50毫米三盐酸 (pH 7.4), 180 毫米氯化钠, 20% 甘油, 0.2% NP-40 和1x 蛋白酶抑制剂鸡尾酒</td>
			<td>在使用前立即加入蛋白酶抑制剂鸡尾酒。</td>
		</tr>
</tbody></table>表 1: 解决方案的准备工作。

Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

在这里, 我们描述了一个共同免疫沉淀协议, 研究在缺氧条件下内源核蛋白的蛋白质相互作用。该方法适用于低氧转录因子与转录联合调节剂相互作用的证明。

缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

co-immunoprecipitation-assay-using-endogenous-nuclear-proteins-from

Nanyang Technological University

Research

JoVE Journal

Biology

17.1K 次观看.  Nanyang Technological University. 在这里, 我们描述了一个共同免疫沉淀协议, 研究在缺氧条件下内源核蛋白的蛋白质相互作用。该方法适用于低氧转录因子与转录联合调节剂相互作用的证明。

视频: Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

神经科学

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

Source: Fuchs, A. R.,et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

染色质免疫沉淀测定:一种研究细胞中蛋白质-DNA 相互作用的技术

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...