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Catalase from bovine liver


Company: Sigma-Aldrich
Catalog#: C3155
Other protocol()

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
[Abstract]  Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and ... [摘要]  通过激动剂配体激活G蛋白偶联受体(GPCR)是通过从无活性受体构象向活性受体构象的转变来介导的。我们描述了一种新颖的单分子测定法,可以实时监测单个GPCR分子中的激活连锁构象转换。受体在跨膜螺旋6末端用Cy3荧光探针进行位点特异性标记,并在连接到显微镜载玻片的磷脂纳米圆盘中重构。然后通过单分子全内反射荧光显微镜随时间监测单个受体分子,显示无活性和活性样构象之间的自发转变。该测定提供关于无活性和活性受体构象的平衡分布以及构象交换的速率常数的信息。实验可以在不存在配体的情况下进行,显示负责基础信号传导活动的自发构象过渡,或者存在激动剂或反向激动剂配体,揭示配体如何改变受体的动力学刺激或抑制信号传导活性。所得到的机械信息对于改进的GPCR靶向药物的设计是有用的。单分子测定法在β2肾上腺素能受体的背景下进行了描述,但可扩展到多种GPCRs。
【背景】GPCR介导本地和远距离的细胞通讯,特别是内分泌系统。例如,细胞对激素如肾上腺素的反应是通过肾上腺素能受体介导的,其中β2肾上腺素能受体(β2AR)是突出的成员。 ...

Single Molecule RNA FISH in Arabidopsis Root Cells
[Abstract]  Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al ... [摘要]  允许研究基因表达调控的方法不断前进。在这里,我们提出了一种能够检测植物根细胞中单个mRNA分子的原位杂交方案,从而允许mRNA在固定样品内的准确定量和定位(Duncan等人, ,2016; Rosa等人,2016)。这种单分子RNA荧光原位杂交(smFISH)使用多个单标记寡核苷酸探针结合靶RNA并产生可以使用宽场荧光显微镜检测的衍射限制信号。我们调整了该方法的最新版本,该方法使用48个荧光标记的DNA寡核苷酸(20个)与每个转录物的不同部分杂交(Raj等人,2008)。这种方法简单易行,具有很好的应用于任何遗传背景的优点。


Detection and Measurement of ROS in Tobacco Leaves
[Abstract]  Leaf metabolism produces hydrogen peroxide (H2O2) at high rates, high level H2O2 accumulation can cause oxidative stress. This protocol describes a method for determining H2O2 concentration in tobacco leaves. In this method all extractions were performed with HClO4, neutralized, and pretreated with ascorbate oxidase to eliminate ascorbate interferences. H2O2 content was determined using a colorimetric assay spiked with an internal control. Interfering peroxides were determined in parallel using a negative control treated with catalase and subsequently subtracted. [摘要]  叶代谢以高速率产生过氧化氢(H 2 O 2 O 2),高水平的H 2 O 2 O 2积累 可引起氧化应激。 该方案描述了用于测定烟草叶中H 2 O 2 O 2浓度的方法。 在该方法中,所有提取用HClO 4进行,中和,并用抗坏血酸氧化酶预处理以消除抗坏血酸盐干扰。 使用掺入内部对照的比色测定法测定H 2 O 2含量。 使用用过氧化氢酶处理的阴性对照平行测定干扰过氧化物,随后扣除。