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Glutaraldehyde solution


Company: Sigma-Aldrich
Catalog#: G6257
Other protocol()

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
[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 phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition. [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。

Cytohistochemical Determination of Calcium Deposition in Plant Cells
[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 was adapted from former reports (Stockwell and Hanchey, 1982; Borgers et al., 1982). The principle of this protocol is that the Ca2+ can react with antimonite and from black ... [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。

Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves
[Abstract]  A technique of atomic force microscopy (AFM) called PeakForce quantitative nanomechanical mapping (PeakForce QNM) is an efficient tool for the quantitative mechanobiological imaging of fibrillar aggregate, human epidermal cell and woody plant cell wall topography (Sweers et al., 2011; Heu et al., 2012; Ďurkovič et al., 2012; Ďurkovič et al., 2013). Here, we describe a detailed protocol for the measurement of nanomechanical properties of primary xylem cell walls in woody plants, for the determination of reduced Young’s modulus of elasticity (MOE), adhesion, deformation, and energy dissipation (Figure 1). This new technique provides direct control of the maximum loading force and the deformation depth in cell wall samples keeping indentations small, while ... [摘要]  称为PeakForce定量纳米机械映射(PeakForce QNM)的原子力显微镜(AFM)技术是用于纤维聚集体,人表皮细胞和木本植物细胞壁形貌的定量机械生物学成像的有效工具(Sweers等人, 2011; Heu等人,2012;Ďurkovič等人,2012;Ďurkovič等人,2013年)。在这里,我们描述一个详细的协议,用于测量木本植物中主要木质部细胞壁的纳米机械性能,用于确定降低的杨氏弹性模量(MOE),粘附,变形和能量耗散(图1)。这种新技术提供了对细胞壁样品中的最大负载力和变形深度的直接控制,保持压痕小,同时消除损伤侧向力,以便保持AFM尖端和植物样品。高分辨率和非破坏性成像为木质植物细胞壁的结构生物学提供了新的定量机械的见解。该程序还可适用于具有变化的刚度范围的其他生物样品。