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Company: AppliChem
Catalog#: A1379
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Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
Author:
Date:
2017-03-05
[Abstract]  Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana ... [摘要]  自从发现CRISPR(聚集的定期交织的短回文重复) - 相关蛋白(Cas)作为植物基因组编辑的有效工具(Li等人,2013; Shan等人已经出版了诸如基因敲除,敲入或转录调控等各种各样的应用,例如,2013; Nekrasov等人,2013)。到目前为止,植物中多种突变体的产生涉及繁琐的杂交或诱变,随后大量人群的耗时筛选,Cas9系统的使用似乎是有希望的方法来克服这些问题。我们设计了一种二元载体,其结合了在拟南芥UBIQUITIN10(UBQ10)启动子和引导RNA(gRNA)控制下的优化的化脓性链球菌(Caspase)密码子的编码序列)由 A驱动的表现盒。拟南芥U6 - 启动子,用于在拟南芥中进行有效的多重编辑(阎等人,2016年)。在这里,我们描述了一个逐步的方案,以经济有效的方式生成含有多个gRNA的二元载体和基于经典克隆方法的Cas9核酸酶。背景 RNA引导的Cas9系统源于针对外源DNA的细菌防御系统(Sorek等人,2013)。由于其高效率,易于处理和多重编辑的可能性,已经被认为是基因组编辑的选择方法。通常,Cas9基因编辑系统涉及单个合成RNA分子,其指导Cas9蛋白质靶向所需DNA位点以进行基因组修饰或转录控制的gRNA。 gRNA-Cas9复合物通过gRNA-DNA配对识别靶向的DNA,并需要存在原始相邻基序(PAM)。 ...

Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits
Author:
Date:
2016-12-20
[Abstract]  Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques. [摘要]  基于活性的探针(ABP)是不可逆地结合特定酶家族的活性中心并且可以偶联至荧光团或亲和标签的小有机分子(Li等人,2013)。在这里,我们描述了使用生物素化的蛋白酶体特异性ABP生物素 - 环氧丙素(Bio-EP)在细胞裂解物中下拉活性催化标准和免疫蛋白酶体亚基的方法。用Bio-EP共价标记活性催化亚单位,然后使用链霉抗生物素蛋白包被的珠子进行下拉。从珠中洗脱后,可以通过Western印迹,串联质谱法(Li et al。,2013)或其他技术检测富集的亚单位。

背景 蛋白酶体是存在于真核细胞的细胞核和细胞质中的桶形多分子酶复合物。蛋白质降解是重要的,包括加工MHC I呈递的抗原肽,并调节许多细胞过程(Kammerl和Meiners,2016)。在造血起源的细胞中,标准(组成型)蛋白酶体通常被免疫蛋白酶体(Meiners等人,2014)所替代,其在三种不同的催化活性β-亚基的掺入中不同(图1)。
为了研究单个催化亚基的分子功能并调节生理过程,亚基特异性蛋白酶体抑制剂的发展是必不可少的。 de ...

Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
Author:
Date:
2016-11-05
[Abstract]  Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid metabolism (Denechaud et al., 2016). The protocol is divided in 2 parts. Part I: Glycolysis experiment is assessed using the Seahorse extracellular flux (XF) analyser. Glycolysis is determined via the measurement of the extracellular acidification rate (ECAR) of the media, which come predominately from the cellular excretion of lactic acid after the conversion of glucose in pyruvate. Part II: De novo lipogenesis experiment determines ... [摘要]  需要代谢通量分析来提供细胞中发生的代谢调节的见解。目前的协议描述了确定糖酵解和肝细胞脂肪生成的快速和可重复的方法。肝细胞的原代培养是用于研究肝葡萄糖和脂质代谢的"体外"模型(Denechaud等人,2016)。协议分为2部分。第I部分:使用海马细胞外通量(XF)分析仪评估糖酵解实验。糖酵解通过测量培养基的细胞外酸化速率(ECAR)来确定,所述培养基主要来自丙酮酸中葡萄糖转化后乳酸的细胞排泄。第II部分:新生脂肪生成实验测定来自乙酸盐C 14+前体的甘油三酯(TG)中的放射性C 14+掺入。在2小时后,向培养基中补加乙酸盐,提取脂质,并在新合成的TG标记的定量之前通过TLC(薄层色谱)分离。

[背景] 有不同的方法来评估葡萄糖和脂质代谢:代谢物定量,酶活性和代谢组学...我们的协议聚焦于活细胞的代谢通量分析并且不需要代谢组学设施。海马细胞外通量(XF)分析仪,现在存在于很多机构,是通过确定培养基pH值间接测量活细胞间接糖酵解的强大工具。脂肪生成协议不需要大投资,是高度可重复的。也可以使用氚化水确定,其通过脂肪酸合酶引入新鲜合成的脂质中。

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