{{'Search' | translate}}
 

Centrifuge

Centrifuge

Company: Eppendorf
Catalog#: 5804 R
Bio-protocol()
Company-protocol()
Other protocol()

Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana
Author:
Date:
2020-09-05
[Abstract]  Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are separated during translation by a 2A “self-cleaving” peptide. This approach enables changes in the relative abundance of a target protein to be detected sensitively, reducing variability in expression of the ratiometric reporter transgene that may occur across different tissues or transformation events. We recently developed a set of Gateway-compatible plant transformation vectors termed pRATIO that combine a variety of promoters, fluorescent and ... [摘要]  [摘要] 比率报告基因是动态测量目标蛋白质相对丰度的工具。在这些系统中,与荧光或生物发光报告基因融合的靶蛋白以固定的化学计量比与与第二报告基因融合的参考蛋白表达。两种融合蛋白均编码在单个转录本上,但在翻译过程中被2A“自切割”肽隔开。这种方法使目标蛋白质的相对丰度的变化能够被敏感地检测到,从而减少了可能在不同组织或转化事件中发生的比例报告基因转基因表达的变异性。我们最近开发了一套与网关兼容的植物转化载体,称为pRATIO 结合了多种启动子,荧光和生物发光报告基因以及源自口蹄疫病毒的2A肽。在这里,我们详细描述了如何使用双荧光比率记者pRATIO3212检查靶蛋白的瞬时表达后的相对丰度烟草本塞姆氏叶。对于此示例,我们分析了响应于karrikins 和rac -GR24 处理的拟南芥MAX2 1(SMAX1)蛋白抑制子的降解。该方案提供了一种简单,快速,易于扩展的方法,用于体内分析农杆菌浸润的烟草叶片组织中的相对蛋白质丰度。

[背景] Karrikins (KARs )是在烟雾中发现的丁烯内酯化合物,可刺激拟南芥种子萌发并增强幼苗的光形态发生(Flematti ,2004; Nelson 等,2009、2010 和2012)。拟南芥中的KAR响应需要α/β- 水解酶KARRIKIN对光的敏感性/低敏感性(KAI2 / HTL)(Sun and Ni,2011; ...

In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals
Author:
Date:
2018-08-20
[Abstract]  We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid ... [摘要]  我们使用体内和体外磷-31核磁共振( 31 P-NMR)光谱来跟踪运输,分区和 外生菌根真菌 Hebeloma cylindrosporum 中的磷酸盐代谢响应来自宿主( Pinus pinaster )或非宿主( Zea mays )的根信号植物。 开发了一种用于体内研究的装置,其允许连续氧化的矿物质溶液在含有菌丝体的NMR管中循环。 在几个连续的处理步骤(在液氮中冷冻;用高氯酸压碎;消除高氯酸;冷冻干燥;在适当的液体培养基中溶解)后,对真菌材料进行体外研究。

【背景】 菌根真菌和植物之间的关联改善了宿主植物的P营养(Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith 等人,,2015)。这种积极效应主要归因于真菌菌丝对磷酸盐(Pi)的吸收,探测了在活跃吸收根周围的耗竭区以外的大量土壤(Smith和Read,2008; Cairney,2011; Smith et al。< em="">,2015)和真菌细胞分泌细胞外磷酸酶(Quiquampoix和Mousain,2005)。吸收的Pi部分地掺入磷酸化的代谢物,磷脂和核酸中,并且部分地浓缩成多磷酸盐(PolyP),其中它们构成液泡中的储存池(Ashford 等人,,1994)。该协议详述了一种装置,该装置允许通过 31 ...

α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Author:
Date:
2018-07-20
[Abstract]  Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors. Here we present a simple and reproducible protocol to study the aggregation of α-synuclein in a plate-reader based assay. [摘要]  研究淀粉样蛋白如α-突触核蛋白体外聚集是一种方便和流行的工具,可以获得聚集的动力学见解以及研究因子(例如,聚集抑制剂) 影响它。 这些聚集测定通常利用荧光染料硫磺素T作为淀粉样蛋白原纤维形成的敏感荧光报告物,并且以基于读板器的形式进行,允许同时筛选多个样品和条件。 然而,由于原纤维成核的随机性质和调节因子的多样性,聚集测定通常倾向于较差的再现性。 在这里,我们提出了一个简单和可重复的协议,以研究基于读板器的测定中α-突触核蛋白的聚集。

【背景】内源性蛋白质与淀粉样原纤维的聚集是一种致病过程,与几种疾病相关,例如,神经退行性疾病如阿尔茨海默病(AD)或帕金森病(PD)以及全身性疾病如AL淀粉样变性( Knowles et al。,2014)。通过基于硫磺素T荧光的聚集测定,可以在基于板读者的装置中在体外中概括该过程,从而允许根据各种影响因素研究淀粉样蛋白的聚集动力学。

硫磺素T(ThT)是一种荧光染料,最初用于组织学样本中的淀粉样蛋白原纤维染色,于1959年由Vassar和Culling(Vassar和Culling,1959),其在体外检测和定量淀粉样纤维的应用

目前,硫代黄素T的聚集测定主要在荧光板读数器中进行,其中例如,96条件可以同时测试。由于原纤维成核的随机性质和影响蛋白质聚集的多种因素,这些测定法具有较差的重现性。因此,已经采用了增加ThT测定的再现性的策略,例如在测量期间使用井板的轨道摇动以及向孔中添加玻璃珠以改善混合(Giehm和Otzen,2010)。 ...

Comments