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2-Mercaptoethanol

2-巯基乙醇

Company: Sigma-Aldrich
Catalog#: M3148
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Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
Author:
Date:
2018-04-05
[Abstract]  We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the phosphorylation of a transcription factor–that can be visualized through antibody staining. Our devices aim at mimicking conditions in dense tissues, such as lymph nodes. A small number of secreting cells is deposited on a polylysine-coated glass and covered by multiple layers of cytokine-consuming. The cells are left to communicate for 1 h, after which the top layers are removed and the bottom layer of cells is antibody labeled for the response to cytokines. ... [摘要]  我们在这里描述了一种可视化细胞因子分泌细胞周围细胞因子浓度场的方法。主要挑战是生理细胞因子浓度可能非常低,在微摩尔浓度范围内。由于目前不可能直接测量这样的浓度,我们依赖于细胞对细胞因子的反应 - 转录因子的磷酸化 - 可以通过抗体染色显现。我们的设备旨在模仿密集组织中的条件,如淋巴结。少数分泌细胞沉积在聚赖氨酸包被的玻璃上并被多层细胞因子消耗覆盖。将细胞连通1小时,之后去除顶层,并且细胞的底层被抗体标记为对细胞因子的应答。然后通过标准荧光显微镜观察细胞因子场的横截面。这篇手稿总结了我们的方法,以量化密集细胞体外细胞因子介导的细胞间通讯的程度。

【背景】哺乳动物的免疫系统已经发展到能够识别和限制潜在病原体的传播,同时使由免疫系统本身造成的附带组织损伤最小化。为了实现这一点,免疫细胞依赖细胞因子介质网络,这些细胞因子介质能够进行细胞间通讯并广播关于致病性侮辱的大小和性质的信息。大量不同细胞因子与其同源受体强烈结合,通常在纳摩尔或皮摩尔范围内具有特征性结合亲和力。通过细胞因子通讯产生免疫龛。例如,在骨髓和胸腺中,通过基质细胞分泌的白细胞介素-7(IL-7)分别支持增殖的B细胞和T细胞祖细胞的存活(Tokoyoda et al。, 2004; Alves等人,2009)。细胞因子生态位的大小控制成熟祖细胞的数量,从而保持血细胞区室平衡(Böyum,1968; ...

Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP
Author:
Date:
2018-04-05
[Abstract]  GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide. [摘要]  GTP酶是分子开关,在无效GDP结合状态和活性GTP结合状态之间循环。 GTP酶通过其内在的核苷酸交换或通过与鸟嘌呤核苷酸交换因子(GEF)的相互作用来交换核苷酸。 监测体外核苷酸交换,以及与调节蛋白直接相互作用的重构,为GTP酶如何被激活提供了重要见解。 在该协议中,我们描述了使用荧光N-甲基呋喃酰基(MANT) - 鸟嘌呤核苷酸来监测核苷酸交换的核心方法。

【背景】GTPase是鸟嘌呤核苷酸结合蛋白,调节细胞过程的广度,从蛋白质生物合成到细胞周期进展,从细胞骨架重组到膜运输。 GTPases可以被认为是分子开关,它在GDP结合“关闭”状态和GTP结合“开启”状态之间循环;在通过GTP的GDP核苷酸交换结合GTP时,GTP酶变得活跃并且将结合下游效应蛋白以招募和激活这些效应子的生物学功能。 GTP酶通过与开关I环(G2结构域)的高度保守苏氨酸和开关II环(G3结构域)的DxxG基序内的甘氨酸的相互作用结合GTP的γ-磷酸。 GTP水解后,与γ-磷酸相互作用的丧失导致动态构象变化,从而使GTPase变为关闭状态(Vetter and ...

Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
Author:
Date:
2018-03-20
[Abstract]  Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes. [摘要]  核小体是真核染色体包装的基本单元,由围绕核心组蛋白H2A,H2B,H3和H4中的每一个的两个分子包裹的147bp DNA组成。 核小体是对称的,一个对称轴以H3分子的C-末端之间的同源二聚体相互作用为中心。 为了探索核小体对称性的功能性后果,我们设计了一对特异性H3异二聚体,称为H3X和H3Y,使我们能够比较具有单一或双重H3改变的细胞。 我们对异二聚体X-Y相互作用的生物化学验证包括使用二甲基琥珀三酸酯(DMS)进行的核内H3交联。 在这里,我们提供了使用DMS来分析酵母核小体的详细方案。

【背景】组蛋白的翻译后修饰影响染色体生物学的各个方面,包括转录,复制,修复和重组。因为核小体包含每个核心组蛋白的两个拷贝,所以修饰可以是对称的(在两个H3尾部上的相同修饰,例如,在核小体内的两个H3尾部上的K27me(Voigt等人

对于单个核小体内H3X-H3Y相互作用的生化验证,我们生成了表达细菌生物素连接酶BirA,N-末端V5-标记的H3X和N-末端生物素接受表位标记的H3Y的酵母菌株(Beckett等人, 1999)。 ...

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