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50 ml tubes

50ml 离心管,114x28mm,PP

Company: SARSTEDT
Catalog#: 62.547.004
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Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses
Author:
Date:
2020-10-05
[Abstract]  Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion ... [摘要]  [摘要]遗传编码的生物传感器是强大的工具为离子和代谢物的定量可视化在体内。设计和优化此类生物传感器通常需要分析大量变体。体外确定的传感器特性,例如底物特异性,亲和力,响应范围,动态范围和信噪比,对于评估体内数据很重要。该协议为新设计的传感器的体外结合测定提供了可靠的方法。这里我们提出了一个详细的协议用于纯化和体外表征的遗传编码的传感器,例示的His亲和标记的GO-(绿橙色)MatryoshCaMP6s钙传感器。GO-Matryoshka传感器基于在感兴趣的结合蛋白内一步插入一个包含两个嵌套荧光蛋白,圆形排列的荧光绿色FP(cpGFP )和Large Stoke Shift LSSmOrange的盒的方法,从而产生了利用被分析物触发的比例式传感器cpGFP的荧光变化。


[背景技术]将绿色荧光蛋白(GFP)在1962年被鉴定在水母水母维多利亚(下村等人,1962) 。30年后,描述了其首次用作报道基因(Chalfie等,1994)。自从发现以来,GFP变体和其他荧光蛋白为生物科学的主要进步做出了巨大贡献,并且现在已成为生物医学研究中的常用工具(Frommer等,2009)。

各种荧光蛋白(FP)和FP变异体已被用作报道分子或与所有生命王国的生物体中的蛋白融合(Chudakov等,2010 ;Valeur和Berberan- ...

Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates
Author:
Date:
2018-10-05
[Abstract]  Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein’s native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting. [摘要]  蛋白质聚集是几种神经退行性疾病的标志,并且与蛋白质体内平衡受损有关。 这种不平衡是由蛋白质天然构象的丧失引起的,最终导致其在细胞内聚集或异常定位。 使用 C。 线虫聚谷氨酰胺疾病模型,我们详细描述了过滤阻滞测定,一种捕获醋酸纤维素膜中蛋白质聚集体的方法,并允许通过免疫印迹进行检测和定量。
【背景】帕金森氏症,阿尔茨海默氏症和多聚谷氨酰胺疾病等神经退行性疾病的一个病理特征是在大脑不同区域存在蛋白质聚集物(Soto,2003; Stroo et al。,2017)。在多谷氨酰胺疾病的情况下,编码序列中谷氨酰胺(CAG)重复的异常扩增扰乱了蛋白质的天然折叠。结果,错误折叠的蛋白质暴露其氨基酸序列的区域,这使得它易于与其他蛋白质聚集,形成大的,不溶的聚集体,这可能妨碍正常的细胞功能(综述于Kuiper 等人 ,2017)。

已经开发了几种用于检测不溶性蛋白质聚集体的方法,包括例如染料结合测定(例如,硫磺素T,刚果红,NIAD-4)和电子显微镜检查。过滤阻滞测定是一种快速而灵敏的方法,可检测和定量体内和体外形成的蛋白质聚集体,包括聚谷氨酰胺(Scherzinger et al。 ,1997; Wanker et al。,1999),α-突触核蛋白(Recasens et al。,2018),和amyloid-beta聚集体(Bieschke et ...

Root Gall Formation, Resting Spore Isolation and High Molecular Weight DNA Extraction of Plasmodiophora brassicae
Author:
Date:
2018-06-05
[Abstract]  Isolation of DNA from obligate biotrophic soil-borne plant pathogens is challenging. This is because of their strict requirement of living plant tissue for their growth and propagation. A soil habitat further imposes risk of contamination from other microorganisms living in close vicinity of the plant roots. Here we present a protocol on how to prepare DNA suitable for advanced molecular analysis on the soil-borne pathogen Plasmodiophora brassicae, a peculiar unicellular plant pathogenic organism, causing disease on Crucifers. First, it is important to grow Brassica or Arabidopsis plants in infested soils below a temperature of 25 °C under moist conditions to promote root gall formation. Root galls should be harvested ahead of initiation of the decomposing ... [摘要]  从专性营养型土壤植物病原体中分离DNA是具有挑战性的。这是因为他们对植物生长和繁殖的严格要求。土壤栖息地进一步增加了居住在植物根部附近的其他微生物污染的风险。在这里,我们提出了一个关于如何制备适用于土传病原体Plasmodiophora brassicae ,一种特殊的单细胞植物致病生物,导致十字花科病的DNA进行DNA分析的方案。首先,在潮湿条件下,在温度低于25°C的条件下,在感染的土壤中种植芸薹属植物或拟南芥属植物对于促进根gall形成是重要的。在分解过程开始之前,不应迟于接种拟南芥或芜菁植株后四或九周,收获根gall。数量减少的土壤生物的休息孢子通过匀浆gall组织的梯度离心获得。用70%酒精和一套不同的抗生素治疗可促进 P。芸苔纯度。基于CTAB的程序允许分离适合大规模平行测序分析的高质量DNA。

【背景】Plasmodiophora brassicae 是一种土壤传播的植物病原体,其在包括拟南芥属的十字花科家族中引起根虫(棒状杆菌)。根肿病对全世界油菜(油菜)和卷心菜的种植有重大影响。 P上。 brassicae 是指定给超级组Rhizaria的一种专性生物营养素(需要一种生长寄主),Rhizaria是研究最少的真核生物组之一(Sierra等人,2016; Sibbald和Archibald, 2017年)。系统发育上, ...

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