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Difco Agar

琼脂,颗粒状

Company: BD
Catalog#: 214530
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Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
Author:
Date:
2017-09-05
[Abstract]  The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is ... [摘要]  P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道(Bartlett等,2014)。由于其P2X受体之间的独特性质,例如大电导孔的形成,P2X7受体已经涉及破坏性疾病如慢性疼痛(North和Jarvis,2013)。然而,P2X7特异性属性的机制仍然知之甚少,部分原因是纯化这种真核膜蛋白是一个挑战。在这里,我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达,并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后,使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15%甘油的缓冲液在4℃下储存至少2个月,并用于各种功能和结构研究(Karasawa和Kawate,2016)。
【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一,并且是广泛疾病如神经退行性疾病,癫痫和神经性疼痛的有希望的新型药物靶点(North和Jarvis,2013; Bhattacharya和Biber, ...

Antibiotic Disc Assay for Synechocystis sp. PCC6803
Author:
Date:
2016-12-20
[Abstract]  This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with the biosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone of cyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivity of the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or its construction would render the mutant strain more ... [摘要]  该协议描述了如何研究蓝藻中的外细胞壁的完整性。 PCC6803使用抗生素。适用于琼脂扩散试验(Bauer等人,1966),其中将浸渍有特定浓度的抗生素的滤纸盘放置在接种细菌的琼脂平板上。我们测试的抗生素,干扰细菌细胞壁的生物合成/功能,将扩散到琼脂中,并在盘周围产生蓝藻生长抑制区。抑制区的大小反映了菌株对抗生素作用的敏感性,例如,在细胞壁内起作用的蛋白质的突变或其构建将使得突变菌株对相应的抗生素。已经证明该方法可用于表型集胞藻(Synechocystis)突变体的表型。 PCC6803缺乏编码Deg蛋白酶的所有三种基因。显示这些不依赖于ATP的丝氨酸蛋白酶的缺失会影响集胞藻细胞的外细胞层(Cheregi等,2015)。

背景 蓝藻属集胞藻PCC6803(以下简称集胞藻6803)是研究光合作用过程的模式生物。虽然其基因组已经在1996年进行了测序,但仍有超过50%的基因编码具有假设或未知功能的蛋白质。三个基因slr1204 ( htrA ), sll1679 hhoA )和 sll1427 编码hEB的丝氨酸蛋白酶(周质蛋白质降解)家族;尽管进行了详细的分析(参见Cheregi等人,2016和其中的参考文献),它们的准确的亚细胞定位和底物仍然是神秘的。在我们实验室中进行的单个和三重缺失突变体的先前的蛋白质组学和代谢组学表征已经显示出具有在集胞藻6803(Miranda)的外细胞层中或上方的功能的蛋白质的表达改变 ...

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi
Author:
Date:
2016-12-20
[Abstract]  Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique ... [摘要]  活性氧(ROS)是有氧代谢的副产物。尽管作为信号分子的有利方面,ROS主要被认为是作用于核酸,蛋白质和脂质的有害物质。反应性氧物质,特别是过氧化氢(H 2 O 2 O 2),作为防御分子跨越诸如,通过植物为了击败入侵病原体如真菌。营养不良的植物致病真菌本身分泌H 2 O 2 O 2以诱导宿主细胞死亡并促进感染。过氧化氢在一定程度上可以通过膜自由扩散。为了能够监测真菌中的细胞内过氧化氢动力学,我们最近在丝状植物病原体禾谷镰刀菌(Mentges and Bormann,2015)中建立了多功能的HyPer成像技术。 HyPer由插入到原核H 2 O 2 O 2 - 感觉蛋白OxyR的调节结构域(RD)中的循环置换的黄色荧光蛋白(cpYFP)组成。 OxyR域使传感器高度特异于H 2 O 2 O 2。 HyPer的氧化增加了在488nm激发的cpYFP的荧光,并降低了在405nm激发的荧光,从而促进了比例式读数(Belousov等人,2006)。 HyPer原来是对pH敏感。 HyPer的H 2 O 2 O 2 - 感官域中的单个氨基酸突变使传感器对H 2 O 2不敏感。该记者称为SypHer,作为pH变化的对照。
   通过使用HyPer成像技术,我们可以证明:i)HyPer成像能够特异和准确地检测细胞内H 2 O 2 O 2平衡的快速变化,ii)。 ...

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