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PBS, pH 7.4

PBS,pH 7.4

Company: Thermo Fisher Scientific
Catalog#: 10010023
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Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
Author:
Date:
2017-11-20
[Abstract]  Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a ... [摘要]  高尔基体内的逆行囊泡运输被用来运送和分类高尔基酶到适当的池内位置。建立了一个检测方法来研究无细胞系统中囊泡靶向的分子细节。生成了稳定的细胞系,其中反式 - 高尔基酶半乳糖基转移酶(GalT)用CFP或YFP标记。考虑到GalT被循环到稳定状态的小池中,含有GalT的囊泡将靶向含有GalT的池内膜。因此从表达GalT-CFP的细胞分离高尔基体膜,而从GalT-YFP表达细胞制备囊泡。在胞质溶胶和能量再生混合物的存在下,在37℃孵育CFP标记的高尔基体和YFP标记的囊泡,与在冰上孵育相比,在混合物的荧光成像后CFP-YFP共定位显着增加。该测定被验证需要能量,蛋白质和生理学重要的运输组分,如Rab GTP酶和保守寡聚体高尔基体系复合物。该测定法可用于调查影响高尔基体运输机器的生理和病理变化,特别是囊泡束缚。

【背景】细胞内囊泡靶向的分子机制对于解释糖基化稳态,神经递质释放,信号受体的调节和营养摄取等方面的解释是重要的(Ungar和Hughson,2003; ...

Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction
Author:
Date:
2017-11-05
[Abstract]  In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step. Under a microscope, these reactions appear as punctate fluorescent spots, indicating successful PLA reaction and suggesting protein-protein interaction between the two antigens. [摘要]  在这个协议中,我们描述近端连接测定(PLA),一种基于抗体的蛋白质相互作用检测方法。 这种方法依赖于个别一抗与两个推定的相互作用蛋白的特异性结合。 一抗需要有不同的宿主。 针对两种宿主的二抗具有与其连接的互补的寡核苷酸部分。 如果两种抗原紧密接近(推测彼此相互作用),则互补的寡核苷酸可以退火,并且荧光核苷酸可以并入单个DNA聚合步骤中。 在显微镜下,这些反应表现为点状荧光斑点,表明成功的PLA反应并提示两种抗原之间的蛋白质 - 蛋白质相互作用。

【背景】近端连接测定法(PLA)是基于抗体的技术,以确定两种蛋白质是否彼此具有40nm。以这种方式检测到的蛋白质可以通过荧光来识别(Ho等人,2012; Banerjee等人,2015)。这使得PLA成为定位蛋白质 - 蛋白质相互作用的极好工具。 Toll样受体(TLR)途径的激活是致病性威胁的先天性免疫应答的重要组成部分。 TLR识别病原体相关分子并诱导信号级联以实现对感染的快速响应。 TLR2和TLR4是TLR家族的两个研究得非常好的成员,它们对不同的刺激有反应。尽管两种受体都响应细菌感染而激活,但只有TLR4响应脂多糖暴露。它们激活一些共享的信号级联,但包括MyD88 / Traf6通路。该途径的诱导包括形成被称为myddosome的信号复合物(Gay等人,2011; Xiong等人,2011; ...

Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
Author:
Date:
2017-10-20
[Abstract]  A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate the memory trace (Liu et al., 2012; Ramirez et al., 2013). Memory appears to be spread over different regions of the brain rather than being localized to one area. Therefore, the methods used to trace memory have the ability to improve our understanding of neuronal circuits. In this protocol, we introduce a doxycycline-inducible expression system to label the specific neurons associated with the original memory trace. [摘要]  存储器跟踪(也称为存储器枚举)被理论化为大脑中物理存储器存储的机制(Silva等人,2009; Josselyn,2010),并且内存跟踪与一个 特定的神经元群体(Liu et al。,2012; Ramirez等人,2013)。 标记和刺激那些神经元将激活记忆痕迹(Liu et al。,2012; Ramirez等人,2013)。 记忆似乎分布在大脑的不同区域,而不是局限于一个区域。 因此,用于跟踪记忆的方法有能力提高我们对神经元电路的理解。 在本协议中,我们引入多西环素诱导表达系统来标记与原始记忆痕迹相关的特定神经元。
【背景】记忆痕迹是记忆被存储为大脑物理或生物化学变化的理论手段(Ryan等人,2015)。在二十世纪初德国动物学家理查德·塞蒙(Richard Semon)制定记忆追踪概念之后,记忆存储的具体过程一直是神经科学领域辩论的一个未解决的话题(Poo et al。,2016)。尽管记忆机制已经成为几十年来的争论焦点,但已经一致认为,特定的神经元被用于记忆的存储(Liu等人,2012; Ramirez等人, ...

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