秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位
Summary
该协议演示了如何在热应激下跟踪蛋白质核易位, 方法是使用绿色荧光蛋白 (GFP) 融合蛋白作为标记, 4 ‘, 6-diamidino-2-苯基吲哚 (DAPI) 染色。DAPI 染色协议快速, 保留了 GFP 和蛋白质亚细胞定位信号。
Abstract
本协议采用绿色荧光蛋白 (GFP) 融合蛋白和 4 ‘、6-diamidino-2-苯基吲哚 (DAPI) 染色法跟踪蛋白质亚细胞定位变化;特别是在热应力条件下的核易位。蛋白质的反应与外部和内部信号相应。一种常见的机制是改变其亚细胞定位。本文介绍了一种跟踪蛋白质定位的协议, 不需要抗体, 放射性标签, 或共焦显微镜。在这篇文章中, GFP 是用来标记的目标蛋白 EXL-1 在C. 线虫, 一个成员的氯离子胞内通道蛋白 (CLICs) 家族, 包括哺乳动物 CLIC4。采用转化与γ辐射相结合的法, 建立了一种集成的转化exl-1::gfp转基因线 (具有启动子和全基因序列), 并稳定地表达了该基因和gfp。最近的研究表明, 在热应力, 而不是氧化应激, EXL-1::GFP 积累在细胞核。将 GFP 信号与细胞核结构和 DAPI 信号重叠, 证实了 EXL-1 亚细胞在应力作用下的定位变化。本协议提出两种不同的固定方法 DAPI 染色: 乙醇固定和丙酮固定。本文提出的 DAPI 染色协议具有快速、高效的功效, 同时保留了 GFP 信号和蛋白质亚细胞定位的变化。此方法只需要使用 Nomarski、FITC 过滤器和 DAPI 过滤器的荧光显微镜。适用于小型实验室设置、本科生研究、高中学生研究、生物技术教室。
Introduction
蛋白质亚细胞定位的变化是反应热应激、饥饿、氧化应激、细胞凋亡、蛋白质磷酸化等内部或外部信号的一种常见机制。例如, 热应力诱导一个 foxo 蛋白成员 DAF-16 核易位1,2和亲凋亡 BCL-2 蛋白出价 translocates 的线粒体后, 接受死亡信号3,4。可以使用各种技术来检测这些更改。西方印迹和生化分离亚细胞结构 (如线粒体或细胞核) 的组合可以很好地实现目标3。然而, 它需要一个特定的抗体对抗蛋白质的兴趣。因此, 一个完善的抗体成为成功的关键。另一种方法是标签不同的亚细胞结构或细胞器与各种标记, 如绿色荧光蛋白 (GFP), 红色荧光蛋白 (RFP), 黄色荧光蛋白 (YFP), 和 mCherry, 同时标签的蛋白质与其他标记的兴趣。然后, 观察他们在共聚焦显微镜下, 以本地化目标5,6。放射性同位素是标记靶蛋白, 然后检测其亚细胞定位7的替代选择。但是, 这种方法需要对放射性废料进行适当的训练和处理。在诸如缺乏特定抗体、缺少适当的标记或 scarceness 等设备的情况下, 需要考虑一种替代方法。为了确定蛋白质核易位, 它是有吸引力的仅标记目标蛋白质与标记和染色核与化学试剂 4 ‘, 6-diamidino-2-苯基吲哚 (DAPI), 因为这只需要常规荧光显微镜。
Immunolabeling线虫具有抗体是挑战, 由于低渗透性的蛋壳或胶原角质层周围的动物。同时, 由于线虫蛋白与脊椎动物的 orthologs 有明显的差异, 一些商业公司提供特定产品的c. 线虫。小实验室很难为自己生成线虫抗体。社区的研究人员经常使用标记蛋白作为标记来显示蛋白质定位或基因表达。本文以 EXL-1::GFP 为例, 在热应力作用下跟踪蛋白质核易位8。将一个集成的平移exl-1::gfp到动物基因组中, 用于稳定地表达gfp融合的基因。研究表明, exl-1在肠道、体壁肌和其他亚细胞结构中表达8。该协议演示如何将蠕虫同步到第四幼虫 (L4) 阶段, 进行热应激实验, 并在常规荧光显微镜下进行 DAPI 染色、乙醇固定、丙酮固定和成像。
Protocol
Representative Results
Discussion
本文提出了一种快速有效的方法来验证蛋白质亚细胞变化从细胞质到细胞核。蛋白表达由 GFP 融合显示, 而核结构由 DAPI 染色验证 (图 3)。由于染色线虫蛋白具有挑战性, 大多数C. 线虫蛋白亚细胞定位的特点是标记它们与标记蛋白, 如 GFP, 拉扎, mCherry, 和其他18,19,20,21. …
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究使用的C. 线虫菌株取自秀丽遗传学中心, 由国立卫生研究院 (P40 OD010440) 提供支持。这项工作得到了 NIH 的支持: 1R03AG048578-01, 纽约市立大学-CCRG 1501 至俊良, 和 PSC-纽约市立大学 66184-00 44 和 67248-00 45 至俊良。我们感谢凯西野蛮-邓恩亲切地分享她的实验室空间。所有荧光图像都收集在皇后学院的核心设施。我们感谢威廉 j. 赖斯对手稿的评论。
Materials
| DAPI | Biotium | 40043 | both Hoechst 33258 and Hoechst 33342 also works well |
| OP50 | CGC | OP50 | https://cgc.umn.edu/ |
| Caenorhabditis Genetics Center (CGC) | https://cgc.umn.edu/ | ||
| Kim Wipe | Kimberly-Clark Corporation | soft tissue | |
| Zeiss AX10 | Zeiss | fluorescence microscope | |
| Axiovision Rel 4.8 | Zeiss | microscope software | |
| AxioCam MR Rev3 | Zeiss | digital camera | |
| Incubator | |||
| Micro centrifuge | |||
| Transparent nail polish gel | |||
| 60 mm petri dishes | |||
| Glass slides | |||
| Glass coverslips | |||
| 1-20 µL pipettor | |||
| 20-200 µL pipettor | |||
| 200-1000 µL pipettor | |||
| 1-200 µL pipet tips | |||
| 200-1000 µL pipet tips | |||
| 1.5 mL microcentrifuge tubes | |||
| Platinum wire worm pick |
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