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Glutaraldehyde solution

戊二醛溶液

公司名称: Sigma-Aldrich
产品编号: G6257
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Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels
Author:
Date:
2018-10-05
[Abstract]  The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive ... [摘要]  环核苷酸调节的离子通道家族包括环核苷酸门控(CNG)和超极化激活和环核苷酸调节(HCN)通道,其在视觉和嗅觉信号传导和心脏起搏活动中起重要作用。在功能上,这些通道已经通过来自非洲爪蟾卵母细胞和哺乳动物细胞中异源表达的蛋白质的电生理学技术进行了广泛的表征。另一方面,这些蛋白质的表达和纯化用于体外生物物理和结构分析是有问题且昂贵的,因此,文献中仅提供关于纯化通道的有限信息。在这里,我们描述了用于将荧光标记的环核苷酸与真核CNG通道的同源物结合研究的方案。此外,我们描述了如何在竞争测定中直接探测未标记的环核苷酸的结合。使用荧光作为配体结合的灵敏探针可减少所需蛋白质的量,并使用标准实验室设备进行快速简便的测量。
【背景】了解蛋白质在分子细节中的功能需要广泛的微观表征。对于配体门控离子通道,需要进行不同的分析以获得有关蛋白质与配体的特异性相互作用,配体结合位点和孔隙之间的通信以及通道特异性特征(如离子通量和失活或脱敏)的信息。属性。与对应于各种功能状态的通道构象的结构数据一起,这允许开发通道功能和调节的完整机械描述。环核苷酸门控(CNG)离子通道是四聚体钾通道,由于它们在嗅觉和视觉信号级联中的功能而特别受关注(Kaupp和Seifert,2002; Craven和Zagotta,2006)。然而,在确定的条件下,纯化的CNG通道的数据非常有限,主要来自单分子力谱(Higgins ...

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
Author:
Date:
2016-10-05
[Abstract]  This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of ... [摘要]  该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物,泛素和帕金蛋白,在保守的丝氨酸65残基(pS65-泛素和pS65-帕金蛋白)上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体,我们没有测试他们在这个测定中的性能,因为,但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法,使用phos-标记凝胶检测pS65-泛素和pS65-帕金。

[背景] 在细胞中, PINK1是稳定和激活的线粒体膜去极化和其他形式的应力,导致线粒体损伤。活化的PINK1磷酸化泛素,其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终,受损的线粒体被自噬噬菌体衔接子识别,并将被蛋白酶体和自噬(mitophagy)降解。这种关键的线粒体质量控制通路促进线粒体的周转,并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
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Cytohistochemical Determination of Calcium Deposition in Plant Cells
Author:
Date:
2016-01-20
[Abstract]  Calcium plays important roles in maintaining plant cellular structure and also acts as a key secondary messenger in intercellular signaling. Thirty years ago, methods of detecting calcium in sub-cellular level had been established (Stockwell and Hanchey, 1982; Borgers et al., 1982) and reviewed extensively (Wick and Heplerm, 1982). We had used the method of testing calcium localization in salt tolerance improved transgenic alfalfa plant (Zhang and Wang, 2015). Here, we describe the protocol of testing calcium deposition by staining with potassium pyroantimonate (PPA) in detail, which ... [摘要]  钙在维持植物细胞结构中起重要作用,并且还在细胞间信号传导中作为关键的第二信使。三十年前,已经建立了在亚细胞水平检测钙的方法(Stockwell和Hanchey,1982; Borgers等人,1982)并广泛地综述(Wick和Heplerm,1982)。我们使用测试钙定位的方法在耐盐改良的转基因苜蓿植物中(Zhang和Wang,2015)。在这里,我们描述了通过用详细的焦锑酸钾(PPA)染色测试钙沉积的方案,其改编自以前的报道(Stockwell和Hanchey,1982; Borgers等人,1982)。该方案的原理是Ca 2+ 2+可以与锑矿和黑色颗粒反应,这可以在透射电子显微镜下观察到。该协议包括植物组织的常见显微操作技术,用透射电子显微镜和照相观察。

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