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Company: Sigma-Aldrich
Catalog#: T1503
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BMV Propagation, Extraction and Purification Using Chromatographic Methods
Author:
Date:
2018-07-20
[Abstract]  Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral capsids in nanotechnology. Herein, we describe a method for BMV purification based on ion-exchange and size-exclusion chromatography. The presented method is of similar efficiency to previously described protocols relying on differential centrifugation and can easily be scaled up. The resulting BMV capsids are stable and monodisperse and can be used for further applications. [摘要]  雀麦花叶病毒(BMV)是众所周知的植物病毒,代表单链RNA(ssRNA)正义病毒。 它已被广泛用作植物病毒生物学,流行病学和病毒衣壳在纳米技术中的应用的多项研究中的模型。 在本文中,我们描述了基于离子交换和尺寸排阻色谱的BMV纯化方法。 所提出的方法与先前描述的依赖于差速离心的方案具有相似的效率,并且可以容易地按比例放大。 得到的BMV衣壳是稳定的并且是单分散的,并且可以用于进一步的应用。

【背景】纳米技术要克服的关键挑战之一是制定有效的和组织特异性的药物递送方法。植物病毒和病毒样颗粒(VLP)具有生物相容性和可生物降解性,不含对人类或动物健康有害的病原体,是合成药物载体的安全替代品,通常会激活免疫系统的不良反应或积聚在免疫系统中。身体到毒性水平。最后,病毒衣壳的生产相对便宜且快速(Ren et al。,2007; Arcangeli et al。,2014)。

Bromoviridae 家族的雀麦花叶病毒(BMV)是用作纳米颗粒载体的良好候选物,因为它显示出所有上述特征并且是研究最多的植物病毒之一(Figlerowicz,2000; Alejska et al。,2005; Urbanowicz et al。,2005; Wierzchoslawski et al。,2006; Kao vet al。 ...

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Author:
Date:
2018-07-05
[Abstract]  Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure ... [摘要]  来自产后啮齿动物的小脑颗粒神经元(CGN)已被广泛用作研究神经元发育,生理学和病理学的模型。 CGN体外培养维持成熟小脑颗粒细胞在体内显示的相同特征,包括发育致密的神经炎网络,神经元活动,神经递质释放和神经元的表达 蛋白质标记。 此外,CGN代表了梭菌神经毒素(CNT)研究的便利模型,最着名的是破伤风和肉毒杆菌神经毒素,因为它们大量表达CNT受体和神经元内基质, ie ,可溶性N-乙基马来酰亚胺 - 敏感因子激活蛋白受体(SNARE蛋白)。 在这里,我们描述了从出生后大鼠/小鼠获得高纯度CGN培养物的方案和用CNT中毒的简便方法。 我们还说明了评估CNT活性及其抑制的方便方法。

【背景】梭菌神经毒素(CNT)的大家族由破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNT)的多种变体形成,它们分别是破伤风和肉毒中毒的神经麻痹毒素(Schiavo et al。,2000; Johnson和Montecucco,2008; Rossetto et al。,2014)。 TeNT,七种BoNT血清型(BoNT / A至/ G)及其许多亚型是金属蛋白酶,通过切割SNARE蛋白(可溶性N-乙基马来酰亚胺敏感因子激活蛋白受体),三种必需蛋白质来阻断神经递质的释放而引起神经麻痹。控制突触小泡与突触前质膜的融合(Rossetto et al。,2014; ...

Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
Author:
Date:
2018-07-05
[Abstract]  The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from Arabidopsis thaliana. [摘要]  不稳定的类囊体蛋白复合物的分级组成和相互作用可以通过连续的2D天然凝胶电泳系统来评估。 温和的非离子洗涤剂洋地黄皂苷用于溶解不稳定的蛋白质超级和巨型复合物,然后用第一维蓝色天然聚丙烯酰胺凝胶电泳(1D-BN-PAGE)分离。 将洋地黄皂苷衍生的蛋白质复合物用更强的去污剂β-DM进一步溶解,随后在正交的2D-BN-PAGE上分离,以从较高级的超复合物中释放较小的蛋白质亚复合物。 在这里,我们描述了来自拟南芥的类囊体蛋白复合物的2D-BN-PAGE分析的详细方法。

【背景】在类囊体膜中发生光合作用的光反应,在高等植物中,由贴壁的grana类囊体和非贴壁的基质类囊体组成。光反应由多亚基蛋白复合物光系统(PS)I和II,细胞色素b 6 f和ATP酶催化。 PSII及其光捕获天线复合物(LHCII)在grana-thylakoids中最为丰富,因此在空间上与基质类囊体定位的PSI-LHCI复合物隔离(Andersson和Anderson,1980)。格拉纳和基质类囊体之间的间期在两个光系统中都得到了丰富(Albertsson,2001; Suorsa et al。,2015)。通过光依赖性LHCII和PSII蛋白的可逆磷酸化介导,光系统与LHCII一起组装成更大的超级和超级复合物。 ...

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