我们感谢 m. 莫拉莱斯和 j. 布拉沃的帮助与设计的南太常委会和 j.c valo 为他的帮助下的下一项协议。我们感谢德尔雷伊的技术援助。这项工作得到了西班牙 Ministerio de Economía y Competitividad 的支持;菲德 BFU2015-70040-R, 西班牙, 卡斯蒂利亚-莱昂菲德 SA026U16 和基金会拉蒙 Areces。Jaraíz-罗德里格斯和冈萨雷斯-桑切斯是来自卡斯蒂利亚·莱昂和欧洲社会基金的奖学金获得者。

所有的实验程序都是在萨拉曼卡大学进行的。
1. 细胞
<ol>
	<li>在开始手术前两天, 将所需密度的细胞板在实验当天汇合。将细胞放入烧瓶或盘子中。然而, 蛋白质提取将更容易从板块。每项实验的每个条件, 都可以方便地准备至少4块 78 cm2或2烧瓶的 150 cm2 , 以确保结果是一致的。</li>
	<li>在本研究中, 板人 G166 GSCs 4 烧瓶的150厘米2, 培养在 RHB-一个干细胞培养基补充 2% B27, 1% N2, 20 ng/毫升 EGF 和 b-细胞生长因子描述的波拉德 et al.22。到达汇合的过程。例如, 当 5 x 106 G166 单元被镀在 150 cm2烧瓶中时, 它们是在电镀后2天处理的。</li>
</ol>
2. 化南太常委会
<ol>
	<li>三十年代在 8200 x g上旋转含有冻干化南常委会 (BCPPs) 的小瓶, 以避免盖子上残留的一些粉末。包括一个控件 BCPP, 以确保找到的交互是特定于目标序列的。在本研究中, 控制 BCPP 是达生物素 (达 b) 和治疗 BCPP 是 TAT-Cx43266-283-b。其他控制可以使用, 如达熔化到混乱或突变的片段结合到生物素。</li>
	<li>将相应培养基中的 BCPPs 溶解到制造商所指示的库存溶液中;例如, 获得2毫克/毫升 BCPP 的股票解决方案, 以治疗 GSCs 添加0.5 毫升的 GSC 培养基到一瓶含有1毫克的 BCPP。涡流, 并确保肽是很好的溶解。</li>
</ol>
3. 管材
注: 7 节所需的每项条件至少准备十二1.5 毫升管。
<ol>
	<li>标记前3管每个条件。它们将在步骤6.4 中获得细胞裂解的总体积。</li>
	<li>标记 A 在每条件3管。这些管子将有第一清获得后, 溶和纺纱细胞裂解, 步骤7.2。</li>
	<li>每条件3根管子上标一 B。这些管子将有一个小分的第一清。这些裂解将用作步骤7.3 中的印迹样本。</li>
	<li>每条件3根管子上标一个 C。这些管子将有清获得后, 下拉 NeutrAvidin, 步骤7.7。 注意: 这是重要的情况下, 西方印迹没有显示蛋白质的信号, 这意味着蛋白质没有被拉下来或失去了在程序的某些步骤。如果是这种情况, 重复的过程, 以拉下来的蛋白质。</li>
</ol>
4. BCPPs 的细胞治疗
<ol>
	<li>吸气培养基。</li>
	<li>根据孵化时间, 将所需的培养基的体积 BCPPs 在尽可能小的介质中, 取代相应的新鲜培养基体积。这是非常重要的, 在任何情况下, 介质完全覆盖板/烧瓶的整个表面, 使细胞不干, 例如, 6 毫升每 150 cm2为30分钟的孵化。</li>
	<li>在细胞培养物中加入 BCPP 的储存液的体积, 以达到被证明有效的浓度。在本研究中, 50 µM TAT-Cx43266-283B 已被证明可以减少 GSC 的增殖。
	<ol>
		<li>因此, 添加92.8 µL 2 毫克/毫升 TAT-Cx43266-283-b (兆瓦 = 3723.34 克/摩尔) 每毫升培养基, 以获得最终浓度50µM TAT-Cx43266-283-b。如果要添加的体积是不同的控制肽, 完成与文化培养基多达相同的最终卷。例如, 49.1 µL 达 b (兆瓦 = 1914.31 克/摩尔) 加上43.7 µL GSC 培养基增加每毫升培养基, 以获得最终浓度50µM 达 b。</li>
	</ol>
	</li>
	<li>将细胞放在37° c 和 5% CO2中, 以确保 BCPP 和其胞内伙伴之间的相互作用发生。如果相互作用需要更长的时间, 孵育时间更长或调整时间, 以防实验由时效。此外, 通过刺激不同的细胞内信号通路, 可以促进或防止兴趣的相互作用。</li>
</ol>
5. 缓冲器和解决方案。
<ol>
	<li>制备 PBS pH 值 7.4: 136 毫米氯化钠;2.7 毫米氯化钾;7.8 mM Na2HPO4· 2H2O;1.7 mM2PO4。</li>
	<li>准备蛋白质裂解缓冲液:20 毫米三盐酸 (pH 8.0), 137 毫米氯化钠, 1% IGEPAL, 使用前, 添加以下: 1/100 (v/v) 蛋白酶抑制剂鸡尾酒, 1 毫米氟化钠, 1 毫米 Phenylmethanesulfonyl 氟化物 (PMSF) 和0.1 毫米钠钒。</li>
	<li>准备 Laemmli 缓冲液: (4x: 0.18 m 三盐酸盐 pH 值 6.8; 5 米甘油; 3.7% SDS (p/五); 0.6 米β-基或9毫米的双制式; 0.04% (v/v) 溴蓝色 (BB))。</li>
</ol>
6. 蛋白质提取
注意: 蛋白质提取是按照先前描述的20,23进行的。在4° c 执行这个过程的整个部分。
<ol>
	<li>完全吸取培养基。</li>
	<li>洗涤 3 x 10 毫升与 ice-cold 磷酸盐缓冲盐水 (PBS) 每 150 cm2非常仔细地避免细胞脱离。</li>
	<li>要获得细胞裂解, 添加3毫升的裂解缓冲每 150 cm2 , 并彻底刮表面使用一个细胞刮板。倾斜的板/烧瓶约45度将使更容易收集细胞裂解到相应的管。</li>
	<li>在三1.5 毫升的管子中, 每管倒入1毫升的细胞裂解液。这些管将被标记的条件和复制, 如步骤3.1 所示。</li>
</ol>
7. 下拉
<ol>
	<li>离心1.5 毫升管在 1.1万x g为10分钟在4° c。</li>
	<li>将清转到新管 (A)。</li>
	<li>每一个条件对不同的管子 (B) 分, 即每管50µL。添加 4x Laemmli 缓冲器 (16.6 µL 50 µL 的裂解液), 并在-20 ° c 冻结。这些裂解将作为通常的西方印迹样品。</li>
	<li>质很好的 NeutrAvidin 琼脂糖由温和的晃动。削减吸管提示, 以增加其直径和改善移的珠子。</li></ol>在 (A) 管中加入50µL NeutrAvidin 琼脂糖每毫升的细胞裂解液。
	<li>在4° c 的夜间孵育, 以非常温和的晃动, 让 NeutrAvidin 与 BCPPs 的细胞内伙伴进行互动。</li>
	<li>离心机在 3000 x g为1分钟在4° c, 以收集 NeutrAvidin 与蛋白质的复合物。</li>
	<li>小心拆卸清, 并将其转移至清洁管 (C)。让他们使用它们, 以防下拉不成功。</li>
	<li>洗涤步骤: 添加新鲜的裂解缓冲颗粒 (管 A), 重的反演和重复步骤 7.6) 和 7.7) 5 次。所有这些清都可以被丢弃。</li>
	<li>卸下清, 并将 2x Laemmli 缓冲器添加到所需的最终音量 (40 µL 每1.5 毫升的管状颗粒从150厘米2的汇合细胞的烧瓶中获得)。</li>
	<li>洗在100° c 为 5 min 离解蛋白质和离心机之间的互作用在三十年代在 8200 x g到药丸 NeutrAvidin 珠子。</li>
	<li>采取清, 包含分离的蛋白质, 与毛细管提示到新的管子 (D)。如果需要重复洗脱步骤, 可以保留珠子。 注: 清 (管 D) 现在已经准备好被装载在西部污点或结冰在-20 ° c。</li>

8. 西部污点
注意: 西方印迹是按照先前描述的24执行的。
<ol>
	<li>在一个 Midi 细胞电泳系统中, 在双三 (4-12%) midigels 上每个车道上的每道样本的负载当量体积。</li>
	<li>Transblot 蛋白质使用干燥的印迹系统到一个硝化棉规则栈。</li>
	<li>用10% 胭脂红将膜染色10分钟。</li>
	<li>冲洗膜 3 x 5 分钟, 5 毫升的 TTBS。</li>
	<li>用7% 的牛奶在 TTBS 1 小时内用管子或小盒子轻轻摇动来阻挡细胞膜。确保所用的容积足以覆盖膜, 并且它们不干燥,即每整个 midi 膜40毫升。</li>
	<li>用5毫升的 TTBS 洗涤 3 x 5 分钟。</li>
	<li>在4° c 的夜间孵育, 用主要抗体对感兴趣的蛋白质进行温和摇动。</li>
	<li>用5毫升的 TTBS 洗涤 3 x 5 分钟。</li>
	<li>在室温下用 TTBS 的过氧化物酶共轭二抗体在1小时内孵育膜。</li>
	<li>用5毫升的 TTBS 洗涤 3 x 5 分钟。</li>
	<li>用化学发光系统中的化学发光基材进行开发。</li>
</ol>
9. 解决问题
<ol>
	<li>如果在开发西部污点以后任何样品没有显示信号在所有为蛋白质, 检查胭脂红。 注: 在胭脂红的膜应显示未定义和众多的乐队沿车道加载。如果带子不是非常引人注目, 装载的蛋白质的数量可能不是足够或转移可能不正确地。考虑重复西方的印迹。</li>
	<li>如果在任何车道的胭脂红膜上都没有污渍, 则从 (C) 管中的步骤7.4 中重复整个过程。</li>
	<li>如果在开发西部污点以后蛋白质信号是非常微弱的, 但胭脂红显示蛋白质, 再孵育膜与主要抗体为更长的时间。如果信号仍然相当微弱, 在室温下再次孵育。</li>
</ol>
10. 本文中使用的其他技术
<ol>
	<li>BCPPs 的免疫细胞化学
	<ol>
		<li>将单元格固定在4% 甲醛 (0.2 毫升/cm2) 中, 用于 20 min。</li>
		<li>洗涤 3x 5 分钟 PBS (0.2 毫升每 cm2)。</li>
		<li>应用相应的抗体 (至少0.15 毫升每 cm2) 稀释抗体溶液在制造商的指示浓度对您的蛋白质的兴趣过夜在4° c。</li>
		<li>在室温下, 用荧光共轭的二次抗体 (每 cm2) 孵育2小时。</li>
		<li>重复步骤 10.1. 2-10.1. 4 与其他抗体对抗你的蛋白质的兴趣。注意不要将相同的物种用于不同的主抗体或相同的波长荧光用于二次抗体。</li>
		<li>使用 antifade 试剂 (每 cm2) 安装单元格 (0.005 毫升)。</li>
		<li>对与数码相机相连的荧光显微镜进行分析。</li>
	</ol>
	</li>
	<li>mtt 法
	<ol>
		<li>培养细胞在37° c 在 24-井板材。</li>
		<li>孵育在黑暗中的细胞75分钟与0.15 毫升培养基每 cm2含有0.5 毫克/毫升 MTT。</li>
		<li>吸入培养基, 在黑暗中孵育10分钟的细胞, 用二甲基亚砜 (0.25 毫升每 cm2) 进行轻度震动。</li>
		<li>使用微读取器测量波长为 570 nm 的吸光度。</li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

有许多方法来研究蛋白质-蛋白质相互作用。本研究提出的方法是基于广泛使用的生物素-素下拉系统, 其中化诱饵孵化与细胞裂解, 允许建立相互作用。本研究中所提出的修正方法包括将该技术与细胞穿透序列相结合。我们建议设计出细胞穿透性的诱饵, 可以用活细胞而不是细胞裂解来孵育, 因此发现的相互作用将反映出细胞环境中发生的那些。
在这里, 我们使用作为细胞穿透序列, 生物素作为下拉标签和 Cx43 的区域组成之间的氨基酸266-283 作为目标, 发现细胞内相互作用的人 GSCs。Cx43 和 c Src 之间相互作用的结构基础是众所周知的11,12。这是一个重要的交互作用, 因为它抑制了胶质瘤细胞的致癌活性24,13。事实上, 南太常委会包含这个区域 (TAT-Cx43266-283) 模仿 Cx43 胶质瘤细胞的 antioncogenic 属性19,20,21。在大鼠胶质瘤 C6 细胞中, TAT-Cx43266-283抑制 c 型 src 的机制包括与其内源性抑制剂沉和 PTEN20一起招募 c-src。应该提到的是, GSCs 是非常有趣的, 作为一个目标, 在胶质瘤治疗, 因为他们构成了一个亚群, 是抵抗常规治疗, 因此负责复发的这种恶性脑肿瘤27。此外, 它们是 hard-to-transfect 细胞, 因此对细胞内相互作用的研究变得更加困难。南南常委会在 GSCs19中迅速而有效地内化, 有利于它们用于细胞内相互作用的研究。在这项研究中, 使用与生物素融合, 我们确认 Cx43266-283序列与 c Src 的相互作用, 连同其内源性抑制剂沉和 PTEN 在人类 GSCs。
该方法对研究生物活性物质的胞内机制具有很强的作用。然而, 确定化细胞穿透饵的生物学效应与 non-biotinylated 的生物作用不同, 是非常重要的。这一步是必须的关联的互动发现的作用, 生物活性化合物。此外, 该化合物的稳定性, 它可能的降解蛋白酶及其可能的毒性, 应仔细测试, 并考虑在计划试验。在所示的示例中, TAT-Cx43266-283对 G166 人 GSCs 的抗效果以前已被记录为20。在本研究中, 我们确认 TAT-Cx43266-283-b 和 TAT-Cx43266-283的抗效果非常相似。此外, 细胞形态学分析显示, α-蛋白和 f-肌动蛋白分布是非常相似的 G166 GSCs 处理 TAT-Cx43266-283-b 或与 TAT-Cx43266-283。总之, 这些结果表明, 在 TAT-Cx43266-283的 c 端包含生物素并没有改变这种化合物对人类 GSCs 的影响。然而, 如果生物素将改变活性分子的作用, 其他标记为蛋白质纯化可以被测试, 例如旗子八 (DYKDDDDK)28, 人流感血来源标记 HA (YPYDVPDYA) 或谷胱甘肽s-转移酶 (GST)29。同样, 如果达不针对细胞的兴趣群体, 其他细胞穿透序列, 如 penetratin, MPG (为审查, 参见30) 或单元格特定序列可以使用31。
除了研究与目标序列具体交互的蛋白质之外, 理想的情况是, 与控制和目标序列相互作用的蛋白质的存在, 以及与它们不相互作用的蛋白质, 如正负控制, 应解决.在这个意义上, 我们发现 Cav-1 在控制和处理的情况下, 表明囊已经参与了内部化的机制, 因为它以前已经显示了26。此外, FAK, 与 c-Src, 但不应与 Cx43 c 终端互动, 在控制和处理的情况下都缺席。这些结果增强了 TAT-Cx43266-283-b、 c Src、沉和 PTEN 之间相互作用的特殊性。为了证实该协议的结果, 共焦显微镜可以用来可视化的分布, 相互作用的蛋白质和研究他们的共存。因此, 我们发现, TAT-Cx43266-283-b 和 c-Src 表现出一个类似的细胞内分布与一些点的共存确认的结果得到了下拉实验。事实上, TAT-Cx43266-283-b 分布在等离子体膜附近, 这意味着在这项研究中, Cx43266-283将该分子定向到其胞内伙伴。
所提出的方法的一个局限性是, 作为诱饵的分子可能无法正确折叠, 并没有发现预期的效果。在这种情况下, 发现的交互不能与效果关联。但是, 这种方法对于信号转导通路中的相互作用尤其有趣, 因为它们通常是由本质上无序的区域32进行的, 因此它们不需要有序折叠。此外, 该方法的优点之一是可以遵循交互的时间过程, 这与瞬态交互尤其相关。此外, 每个残留物对相互作用的相关性可以很容易地研究。事实上, 有可能研究后修饰对蛋白质-蛋白质相互作用的相关性, 例如, 通过 phosphomimetic 取代谷氨酸为丝氨酸或苏氨酸。同样, 用丝氨酸或苏氨酸代替丙氨酸或酪氨酸, 可以检测 non-phosphorylatable 丝氨酸、苏氨酸或酪氨酸的作用.为了模拟磷-酪氨酸, 最准确的方法是 Tyr 对 p-Tyr33的替换。
最后, 这个协议的范围是远远超出蛋白质-蛋白质的相互作用, 因为这个系统可以应用于其他生物活性的货物, 如 RNA 序列, 纳米粒子, 病毒或其他分子, 可以融合到素和转与 CPP 研究他们的细胞内作用机制。

蛋白质与蛋白质的相互作用对于多种细胞过程是必不可少的。为了充分理解这些过程, 需要在复杂的胞内环境中识别蛋白质相互作用的方法。一个最常用的方法来识别蛋白质的相互作用的伙伴是使用该蛋白或模拟肽的一部分, 该蛋白质作为诱饵的亲和力下拉实验后, 检测结合蛋白。由于素和生物素的亲和力、特异性和稳定的相互作用, 素-生物素系统经常被使用,1,2。通常, 生物素是共价键与诱饵 (蛋白质或肽) 和经过一段时间的孵化与细胞裂解允许建立相互作用, 化诱饵绑定到其胞内伙伴是拉下来与素或素衍生物与支撑珠共轭。然后, 在洗涤、洗脱和分析后用变性电泳法检测诱饵蛋白相互作用。这项技术的一个问题是, 兴趣蛋白与细胞内伙伴之间的相互作用发生在细胞环境之外。这对于信号传导通路中的相互作用尤其重要, 因为它们发生在特定的细胞内位置, 它们是瞬态的, 它们通常是由不丰富的蛋白质进行的。因此, 在细胞裂解中, 这些相互作用可以被其他更丰富的蛋白质或通常不接近的蛋白质所掩盖。
细胞穿透肽 (简称南常委会) 是短肽 (≤40氨基酸), 主要由阳离子氨基酸组成, 能够将广泛的分子输送到几乎所有的细胞3。诸如蛋白质、质粒 DNA、siRNA、病毒、成像剂和各种纳米粒子等货物已被共轭到南南常委会并有效地内化4,5。由于这种传输能力, 他们也被称为蛋白质转导域 (PTDs), 膜转运序列 (劳动), 和特洛伊肽。在南南常委会中, 来自 HIV 反蛋白达6的达多肽已成为最广泛研究的7、8、9之一。达是一种 nonapeptide, 含有6的精氨酸和2的赖氨酸残留物, 因此是高阳离子。替代研究表明, 净正电荷是必要的, 与等离子体膜的真核细胞的静电相互作用, 并随后内部化10。同样地, 在其他的南南常委会, 积极带电的达强结合静电的各种消极带电的物种存在于细胞膜的胞外表面, 包括脂质头组, 糖蛋白和蛋白3,10. 由达胞运送的生物活性货物立即免费进入其细胞内伙伴。
在这里, 我们提出了一个方法, 结合了达 CPP 与生物素研究细胞内相互作用。目的是通过将靶生物与生物素融合, 设计出细胞穿透性诱饵。这一建议的主要优点是诱饵和它的合作伙伴之间的相互作用将在其蜂窝环境中进行。为了显示这种方法的有效性, 我们使用了一个小序列的蛋白质 Cx43, 已经报告了互动细胞与 proto-oncogene c Src11,12,13。Cx43 是一种整体膜蛋白, 广泛表达于星形胶质细胞14中, 在高等级胶质瘤中被下调, 是中枢神经系统最常见的恶性肿瘤15、16、17 ,18。先前的研究表明, 与 c-Src (人体 Cx43 中的氨基酸266-283 相互作用的 Cx43 区;Pubmed: P17302) 融合到达 (TAT-Cx43266-283) 抑制 c-Srcin 胶质瘤细胞和胶质瘤干细胞的致癌活动 (GSCs)19,20,21。为了设计胞内诱饵, Cx43266-283已在 N 总站 (TAT-Cx43266-283) 和 C 端 (TAT-Cx43266-283-b) 上的生物素融合到了一起。该策略已成功地应用于大鼠胶质瘤 C6 细胞系中, 以确定 c-src、c-端 src 激酶 (沉) 和磷酸酶和张力同源 (PTEN) 为 Cx43 的这一区域的胞内合作伙伴20。在这里, 我们描述这个方法测试它的功效在人类 GSCs, 这是非常相关的胶质瘤治疗, 但更难染比 non-stem 胶质瘤细胞。

<table>
	<tbody>
		<tr>
			<td>G166 GSC line</td>
			<td>BioRep</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RHB-A stem cell medium</td>
			<td>Takara</td>
			<td>Y40001</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Invitrogen, Life Technologies, ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td>10 &#181;g/ml</td>
		</tr>
		<tr>
			<td>B-27 Serum free Supplement (50X)</td>
			<td>Invitrogen, Life Technologies, ThermoFisher Scientific</td>
			<td>17504-044</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Invitrogen, Life Technologies, ThermoFisher Scientific</td>
			<td>17502-048</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant Human b-FGF</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>PBS pH 7.4: In deionized water, 136 mM NaCl ; 2.7 mM KCl; 7.8 mM Na2HPO4&#183;2H2O ; 1.7 mM KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10 cryopreservation medium</td>
			<td>StemCell Technologies</td>
			<td>7930</td>
			<td></td>
		</tr>
		<tr>
			<td>TAT</td>
			<td>GenScript</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>TAT-B</td>
			<td>GenScript</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>TAT-Cx43266-283</td>
			<td>GenScript</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>TAT-Cx43266-283-B</td>
			<td>GenScript</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Phalloidin</td>
			<td>Molecular Probes, Life Technologies, ThermoFisher Scientific</td>
			<td>A1275737</td>
			<td>1/20</td>
		</tr>
		<tr>
			<td>Monoclonal &#945;-tubulin mouse antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T9026</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Molecular Probes, Life Technologies, ThermoFisher Scientific</td>
			<td></td>
			<td>1.25 mg/ml</td>
		</tr>
		<tr>
			<td>Pierce&#8482; NeutrAvidin&#8482; Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>29200</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein lysis buffer: 5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 2 mM EDTA , 2 mM EGTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Set III. EDTA-Free</td>
			<td>Calbiochem, Bionova</td>
			<td>539134</td>
			<td>1/100 (v/v)</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>PanReac AppliChem</td>
			<td>141675</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Laemmli buffer: (4x: 0.18 M Tris-HCl pH 6.8; 5 M glycerol; 3.7 % (w/v) SDS; 0.6 M &#946;-mercaptoethanol or 9 mM DTT ; 0.04% (v/v) bromophenol blue (BB) .</td>
			<td></td>
			<td></td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Xcell 4 SureLock Midi-Cell Electrophoresis System</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>WR0100</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex Bis-Tris Midi-Gels 4-12%</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>WG1402box</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MOPS SDS Running Buffer (20X)</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>NP0001</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer 20x</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>NP0006</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>iBlot 2 NC Regular Stacks (nitrocellulose membranes)</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>IB23001</td>
			<td></td>
		</tr>
		<tr>
			<td>iBlot 2 Dry Blotting System - Gel transfer device</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>IB21001</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Ponceau S Solution (0.1% Ponceau (w/v) in 5% acetic acid (v/v)) in water</td>
			<td>Sigma</td>
			<td>P7170</td>
			<td></td>
		</tr>
		<tr>
			<td>FAK polyclonal rabbit antibody</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>AHO0502</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Src polyclonal rabbit antibody</td>
			<td>Cell Signalling (WERFEN)</td>
			<td>2108S</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Csk polyclonal rabbit antibody</td>
			<td>Cell Signalling (WERFEN)</td>
			<td>4980</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>PTEN polyclonal mouse antibody</td>
			<td>Cell Signalling (WERFEN)</td>
			<td>9552</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Caveolin-1 polyclonal rabbit antibody</td>
			<td>Abcam</td>
			<td>ab2910</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP antibody</td>
			<td>Quimigen, Santa Cruz Biotechnology</td>
			<td>Sc-2005</td>
			<td>1/5000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-HRP antibody</td>
			<td>Quimigen, Santa Cruz Biotechnology</td>
			<td>SC-2030</td>
			<td>1/5000</td>
		</tr>
		<tr>
			<td>Western Blotting Luminol Reagent</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2048</td>
			<td></td>
		</tr>
		<tr>
			<td>Src polyclonal rabbit antibody</td>
			<td>Cell Signalling (WERFEN)</td>
			<td>2108S</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Antibody solution: PBS, 10% FBS, 0.1 M lysine, 0.02% sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Invitrogen, Life Technologies, ThermoFisher Scientific</td>
			<td>A11029</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Cy2-conjugated streptavidin</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-220-089</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>MicroChemi Luminescence system</td>
			<td>DNA Bio-Imaging Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Apoptosis Kit with Annexin V Alexa Fluor&#174; 488 &#38; Propidium Iodide (PI)</td>
			<td>Molecular Probes, Life Technologies, ThermoFisher Scientific</td>
			<td>V13241</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>SlowFade Gold antifade reagent</td>
			<td>Life Technologies, ThermoFisher Scientific</td>
			<td>S36936</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide (MTT)</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide for UV-spectroscopy, &#62;=99.8% (GC)</td>
			<td>Honeywell</td>
			<td>41641-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Appliskan 2001</td>
			<td>Thermo Electron Corporation, Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

biotinylated cell-penetrating peptides to study intracellular protein-protein interactions

在这里, 我们提出了一个协议来研究细胞内蛋白质-蛋白质相互作用, 是基于广泛使用的生物素-素下拉系统。所提出的修改包括该技术与细胞穿透序列的结合。我们建议设计出细胞穿透性的诱饵, 可以用活细胞而不是细胞裂解, 因此发现的相互作用将反映出在细胞内环境中发生的那些。Connexin43 (Cx43), 一种形成缝隙连接通道和 hemichannels 的蛋白质在高等级胶质瘤中被下调。由氨基酸266-283 组成的 Cx43 区是抑制胶质瘤细胞致癌活性的罪魁祸首。在这里, 我们使用作为细胞穿透序列, 生物素作为下拉标签和 Cx43 的区域组成之间的氨基酸266-283 作为目标, 发现细胞内相互作用的 hard-to-transfect 人脑胶质瘤干细胞。所提出的方法的一个局限性是, 作为诱饵的分子可能无法正确折叠, 因此, 发现的相互作用不能与影响。然而, 这种方法对于信号转导通路中的相互作用尤其有趣, 因为它们通常是由固有无序的区域进行的, 因此它们不需要有序折叠。此外, 该方法的优点之一是可以很容易地研究各残留物在相互作用中的相关性。这是一个模块化系统;因此, 其他的细胞穿透序列, 其他标记, 和其他的胞内靶可以使用。最后, 该协议的范围远远超出蛋白质-蛋白质的相互作用, 因为这个系统可以应用于其他生物活性的货物, 如 RNA 序列, 纳米粒子, 病毒或任何分子, 可以转与细胞穿透序列和融合到下拉标签研究他们的细胞内作用机制。

在使用 BCPPs 研究细胞内相互作用之前, 比较 BCPP 与 CPP 对 BCPP 结果的影响是非常关键的。因此, 为了研究生物素的包含是否改变了目标序列的活性, 我们首先分析了 TAT-Cx43266-283-b 与 TAT-Cx43266-283相比对 G166 GSCs 形态学的影响。为此, 我们进行了一些免疫荧光分析的两个骨架蛋白, F 肌动蛋白和α-蛋白后24小时的治疗。图 1显示, G166 GSCs 在50µM TAT-Cx43266-283或 TAT-Cx43266-283b 之间, 与控件中显示的细长和扩展的蜂窝 prolongations 相比, 获得更圆的形状 (达或乙)。事实上,图 1b显示, 当细胞被 TAT-Cx43 的266-283或 TAT-Cx43266-283B 处理, 而它们在控制单元中形成更多的肌动蛋白束时, 肌动蛋白丝主要组装成肌动蛋白网络 (用达或达 B)25。相反, α-蛋白分布在不同的条件之间没有变化。结果表明, 生物素的存在并没有改变靶序列对 G166 GSCs 形态的影响。在以前的研究20,21中, 我们显示了 TAT-Cx43266-283减少了 G166 GSCs 的增殖。在本研究中, 我们研究了 TAT-Cx43266-283-b 在增长中是否与 TAT-Cx43266-283的效果相同。为此, 我们用 MTT 法分析了 72 h 治疗后的 G166 GSCs 增殖。mtt 法是一种评价细胞代谢活性的比色法。MTT 氧化酶在线粒体中代谢, 反映出存活细胞的数量。图 2显示, 当单元格处理时使用50µM TAT-Cx43266-283或50µM TAT-Cx43266-283B 时, G166 GSCs 单元格的生存能力的降低不会有明显差异。事实上, 与对照组相比, 两种 G166 GSCs 增殖都明显减少。
一旦我们确认, 我们的目标序列在 G166 GSCs (TAT-Cx43266-283) 中的作用没有被包含在 C 末端 (TAT-Cx43266-283-b) 的生物素所改变, 我们调查了这个序列的胞内伙伴按照本研究中描述的协议 (图 3)。由于囊参与了达内化的机制26, 我们分析了 caveolin-1 (Cav-1) 在拉下的存在。西方印迹分析 (图 4) 表明, 达 b 和 TAT-Cx43266-283-b 与 Cav-1 交互。然而, TAT-Cx43266-283-b 的能力, 以招募 c-Src, PTEN 和沉是强于发现与达 b。黏着性粘附激酶 (FAK) 是一种未显示与 Cx43 相互作用的 c 型 Src 基板。实际上, FAK 没有显示与 b 或 TAT-Cx43266-283-b 的任何重要交互。
为了确认 TAT-Cx43266-283-b 和 c Src 之间的交互作用, G166 GSCs 在50µM TAT-Cx43266-283-b 中孵育30分钟, 并通过共聚焦显微镜 (图5).结果表明, TAT-Cx43266-283-b 的胞内分布与等离子体膜 (磷脂染色示) 相近, 与 c 型 Src 相匹配。实际上, 共存分析揭示了合并图像中的 TAT-Cx43266-283B 和 c Src 之间的共存 (白色) 点。因此, 共焦显微镜研究证实了在本研究中所描述的 BCPP 下拉协议所取得的结果。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56457/56457fig1.jpg"> 
图 1: BCPP 和 CPP 对 GSC 形态学的影响。 G166 GSCs 被镀在一个低密度 (2 x 104细胞/cm2) 和 24 h 后, 他们孵化与50µM 控制 CPP (达), 控制 BCPP (达 B), 治疗 CPP (TAT-Cx43266-283) 或治疗 BCPP (TAT-Cx43266-283-b).a) f-肌动蛋白 (红), α-蛋白 (绿色) 和合并 + DAPI 免疫的同一领域显示 G166 GSCs 形态学。酒吧 = 50 µm. b) f-肌动蛋白免疫显示不同分布的 f-肌动蛋白在 G166 GSCs 孵化后 24 h 与50µm 控制 cpp (达) 或控制 BCPP (达 b) 相比, 50 µm 治疗 cpp (TAT-Cx43266-283) 或 BCPP (TAT-Cx43266-283-b)。条形 = 10 µm.<a href="//ecsource.jove.com/files/ftp_upload/56457/56457fig1large.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/56457/56457fig2.jpg"> 
图 2: BCPP 和 CPP 对 GSC 活性的影响。 G166 GSCs 被镀在5500细胞/cm2在 24-井板和孵育50µM 控制肽, cpp (达) 或 BCPP (达 B), 或50µM 处理肽, cpp (TAT-Cx43266-283) 或 BCPP (TAT-Cx43266-283-b)。用 MTT 法分析72小时后的细胞活力。结果被表达为 MTT 吸收, 是平均± s.e.m. 的至少3实验 (++ p˂0.01 vs 控制. ** p˂0.01, *** p˂0.001 vs 达或达 B; one-way 方差分析 withTukey 后测试)。请注意, 南太常委会 vs BCPPs 的效果没有显著差异.<a href="//ecsource.jove.com/files/ftp_upload/56457/56457fig2large.jpg" target="_blank">单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56457/56457fig3.jpg"> 
图 3: 协议图。 一步一步的图解描述的过程, 如 "协议", 从孵化的 BCPPs, 直到洗 BCPPs 和他们的相互作用的蛋白质获得. 1) 孵育培养细胞与 BCPPs 的期望浓度所需时间。2) 在孵化过程中, BCPPs 被内化, 它们与细胞内的伙伴相互作用。3) 用 ice-cold PBS 在冰上三次清洗细胞。4) 溶解细胞提取蛋白质。5) 将电池裂解至导管。6) 在 11000 x g处旋转10分钟, 在4° c。7) 将清转移到新管 (a) 上, 并将裂解的小分作为常规的西方印迹样品在试管 (B) 中处理。8) 重 NeutrAvidin 琼脂糖珠, 并添加50µL 每管 a 使用切割吸管尖端。9) 在4° c 下轻轻摇动12小时, 让 NeutrAvidin 琼脂糖珠与 BCPPs 及其合作伙伴进行互动。10) 在 3000 x g上旋转1分钟, 将珠子与化的诱饵及其相互作用的蛋白质结合在一起。11) 将清转移到新的管道 (C), 并在需要重复的情况下保持使用。12) 用新鲜的裂解缓冲液清洗五次, 重, 旋转1分钟, 在 3000 x g上, 丢弃上清。13) 小心去除所有上清液。14) 在三十年代的 8200 x g上, 添加所需的 4x Laemmli 缓冲器的体积和洗的蛋白质, 在100° c 下为 5 min 15) 旋转。16) 将在上清液中发现的洗蛋白转移到新的管 (D)。17) 将其加载到凝胶上进行免疫印迹分析。<a href="//ecsource.jove.com/files/ftp_upload/56457/56457fig3large.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/56457/56457fig4.jpg"> 
图 4: G166 GSCs 中 TAT-Cx43 的细胞内相互作用的研究, 其次是西印迹. G166 GSCs 被孵育与50µM 达 b 或 TAT-Cx43266-283-b。30分钟后, 细胞被裂解和达 b 或 TAT-Cx43266-283-b 附着在他们的细胞内伙伴被拉下来与 NeutrAvidin 珠。用免疫印迹法对洗蛋白进行了加载和分析, 以研究 FAK、沉、PTEN 和 Cav-1 的水平。请注意, Cav-1 与达 b 和 TAT-Cx43266-283-b、c Src、PTEN 和沉交互作用优先与 TAT-Cx43266-283-b 和 FAK 没有显示任何与 b 或 TAT-Cx43266-283b 的交互. <a href="//ecsource.jove.com/files/ftp_upload/56457/56457fig4large.jpg" target="_blank">单击此处可查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56457/56457fig5.jpg"> 
图 5: 通过共焦显微镜对 G166 GSCs 中的 TAT-Cx43266-283B 细胞内相互作用进行了确认。 G166 GSCs 被孵化与50µM TAT-Cx43266-283-b。30分钟后, 细胞被固定和处理, 以本地化 TAT-Cx43266-283-b 与 Cy2-Streptavidin (绿色), c Src 的免疫荧光 (红色) 和磷脂与蛋白 V (蓝色)。请注意在合并图像中, TAT-Cx43266-283-b 和 c-Src 之间的共存 (白色) 与等离子膜的接近点。<a href="//ecsource.jove.com/files/ftp_upload/56457/56457fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions at JoVE.com

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

这是一项研究细胞内蛋白-蛋白质相互作用的协议, 基于生物素-素下拉系统, 具有新颖性的单元穿透序列结合。主要的优点是, 目标序列是用活细胞来培养的, 而不是细胞裂解, 因此相互作用将发生在细胞环境中。

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

biotinylated-cell-penetrating-peptides-to-study-intracellular-protein

Research

JoVE Journal

Biochemistry

9.4K 次观看.  Universidad de Salamanca. 这是一项研究细胞内蛋白-蛋白质相互作用的协议, 基于生物素-素下拉系统, 具有新颖性的单元穿透序列结合。主要的优点是, 目标序列是用活细胞来培养的, 而不是细胞裂解, 因此相互作用将发生在细胞环境中。

视频: Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

光学镊子研究翻译调控中的RNA-蛋白质相互作用

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...