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Methanol

甲醇

Company: Sigma-Aldrich
Catalog#: 322415
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Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts
Author:
Date:
2020-11-20
[Abstract]  

With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary

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[摘要]  [摘要]随着CRISPR-Cas的出现以及能够轻松修饰各种生物的基因组的能力,越来越多地开发大鼠模型来询问乳腺发育和肿瘤发生的遗传事件。已经为小鼠和人类组织彻底开发了用于分离和表征乳腺上皮细胞亚群的方案,但是对大鼠特异性方案的需求却在不断增长。迄今为止,还没有用于分离大鼠乳腺上皮亚群的标准方案。分析大鼠乳腺层次的变化将有助于我们阐明乳腺癌中的分子事件,乳腺癌亚型的起源细胞以及肿瘤微环境的影响。在这里,我们描述为1)大鼠乳腺上皮细胞分离开发的几种方法;2)大鼠乳腺成纤维细胞分离;3)培养大鼠乳腺上皮细胞;通过4)流式细胞仪分析和鉴定大鼠乳腺细胞;5)免疫荧光。源自该协议的细胞可用于多种目的,包括RNAseq ,药物研究,功能测定,基因/蛋白质表达分析和图像分析。


[背景]大多数与乳腺有关的研究都是在小鼠模型和人体样品中进行的。然而,由于其具有类似于人的药代动力学特征和乳腺发育,该疾病的大鼠模型正变得越来越流行(Russo等人,1990;Jiunn等人,2008; Smalley等人,2016)。像人类腺癌一样,大鼠乳腺癌也经历组织学发展阶段(Russo等,1990; Singh等,2000),并且是卵巢激素依赖性的(Thompson等,1998; ...

Paper Lateral Flow Biosensor for Nodavirus Reverse Transcribed RNA Detection
Author:
Date:
2020-08-05
[Abstract]  Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when ... [摘要]  [摘要] 纸纳米生物传感器已经成为分析导致各种疾病的兽医和人类病原体的一个极好的平台。尤其是侧向流分析或生物传感器是实验室设备和现场分析中灵敏、快速、可靠和准确分析的理想选择。病毒RNA检测对公共卫生和动物健康保护具有重要意义。在这一方面,本协议的重点是开发功能化金纳米粒子侧流生物传感器,用于鱼类神经坏死病毒(Nodavirus)核酸检测。从鱼类标本中分离出的总病毒RNA进行逆转录PCR扩增,扩增产物与特异性寡核苷酸探针混合。当有nodavirus产物存在时,形成红色检测线。这种方法对基础研究有很大的意义,因为它消除了耗时、繁琐的电泳程序的需要,并且可以在养鱼场对养鱼户进行调整。利用这种生物分析平台进行病害监测,无需耗费大量时间和成本,对水产养殖和环境安全有很大影响。

[背景] 关注点和/或现场生物分析一直是关注人类和动物福祉的研究工作的最终目标。基于纸基的传感平台具有功能化简单、重现性好、制造成本低等优点,是一种极具吸引力的分析平台。纸基分析设备已应用于小分子、蛋白质和各种核酸的分析(Parolo和Merkoçi,2013;Bahadir和Sezgintürk,2016;Jiang等人,2019)。侧流生物传感器(LFB)是一种带有干试剂的预制材料条带,通过流体样品激活。它们专为一次性一次性使用而设计,只要有足够的开/关信号(Posthuma ...

Expression and Ni-NTA-Agarose Purification of Recombinant Hepatitis C Virus E2 Ectodomain Produced in a Baculovirus Expression System
Author:
Date:
2018-10-05
[Abstract]  In this protocol, we describe the production and purification of the ectodomain of the E2661 envelope protein (amino acids 384-661) of the Hepatitis C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotic and eukaryotic systems but in small quantities or without native protein characteristics. In our case, we use the Baculovirus expression system in insect cells. E2661 is secreted into the extracellular medium and purified by means of affinity chromatography a Ni-NTA-column because the protein has a tag of six histidines at its amino terminal end. The purified protein possesses a native-like conformation and it is produced in large quantities, around 5-6 mg per liter. [摘要]  在该协议中,我们描述了丙型肝炎病毒的E2 661 包膜蛋白(氨基酸384-661)的胞外域的产生和纯化,其在病毒进入中起基础作用。 进入宿主细胞。 该蛋白质已经在原核和真核系统中表达,但是少量或没有天然蛋白质特征。 在我们的例子中,我们在昆虫细胞中使用杆状病毒表达系统。 E2 661 被分泌到细胞外培养基中并通过亲和层析Ni-NTA-柱纯化,因为该蛋白质在其氨基末端具有六个组氨酸的标签。 纯化的蛋白质具有天然样构象,并且大量生产,每升约5-6mg。
【背景】丙型肝炎病毒(HCV)是全世界慢性肝炎,肝硬化和肝细胞癌的主要原因(Major et al。,2001; Alter,2006)。此时,没有HCV疫苗,抗病毒药物用于治疗HCV感染(Imran et al。,2014)。然而,治疗费用昂贵且不是100%有效(Kohli et al。,2014)。 HCV包膜糖蛋白E2负责与细胞受体的相互作用,因此它是研究病毒感染周期的第一步的主要候选者。由于糖基化和聚集,先前的表达系统产生低水平的异质蛋白质,并且难以区分经历生产性和非生产性折叠的分子(Flint ...

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