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DL-Dithiothreitol

DL-二硫苏糖醇

Company: Sigma-Aldrich
Catalog#: 43815
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Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
Author:
Date:
2018-10-05
[Abstract]  Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to ... [摘要]  在不同细胞区室之间穿梭蛋白质控制它们的蛋白质稳态,并且在某些情况下可以有助于调节它们的活性。 染色质结合蛋白(例如转录因子)的生化分析通常是困难的,因为它们的产率低并且由于核酸的干扰。 该协议描述了一种有效分离细胞的方法,结合机械(即,超声处理)或酶处理(即,benzonase),有助于分析染色质结合蛋白提取物 通过蛋白质印迹分析或蛋白质下拉分析。 该方法对于富集特定亚细胞级分内的特定蛋白质以研究特定的翻译后修饰模式或鉴定特定的蛋白质 - 蛋白质相互作用可能是有价值的。
【背景】许多染色质结合蛋白的活性和翻译后调节研究很少,因为在分离它们进行生化分析时存在技术困难。这甚至是转录因子的情况,例如基本的螺旋 - 环 - 螺旋(bHLH)转录因子,其通常在组织或细胞模型中具有稀缺的时间和空间表达模式(Dennis 等。,2018)。当生物材料的量成为研究分子途径的障碍时,协议细化有助于解除技术限制(Gillotin和Guillemot,2016)。在我们最近的研究中,我们努力了解神经元bHLH转录因子Ascl1的蛋白水解是如何在神经元分化的细胞模型中调节的(Gillotin et ...

MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
Author:
Date:
2017-12-05
[Abstract]  Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides is thus an important task in systems biology, cell biology, biochemistry, and drug design. However, this task is often hampered by the drawbacks of classical biophysical methods for analysis of molecular interactions like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), which require immobilization of the interaction partners or very high sample concentrations. MicroScale Thermophoresis (MST) is an innovative method ... [摘要]  蛋白质 - 肽相互作用是许多生理过程的一部分,例如表观遗传学,其中组蛋白复合物的肽区域对于染色质结构的调节是至关重要的。短肽通常也被用作小分子药物靶向蛋白质复合物的替代物。研究蛋白质和肽之间的相互作用是系统生物学,细胞生物学,生物化学和药物设计中的重要任务。然而,这一任务往往受到经典生物物理学方法分析分子间相互作用的缺陷的困扰,例如表面等离子体共振(SPR)或等温滴定量热法(ITC),其需要固定相互作用配偶体或非常高的样品浓度。 MicroScale热泳(MST)是一种创新的方法,可以确定分子间相互作用的重要参数,如解离常数,化学计量和热力学。而且,它可以快速准确地进行,可以自由选择缓冲液或生物液体,不需要固定样品,样品消耗也非常少。这里我们详细描述了两个MST测定法,其分析(i)真核RNA聚合酶II的某些肽段与真核转录延伸复合物的蛋白质亚基之间的相互作用和(ii)N-末端组蛋白尾肽与表观遗传学之间的相互作用读者蛋白质。这些实验表明,MST能够表征蛋白质 - 肽相互作用,这些相互作用仅由肽的微小变化触发,例如,在特定的丝氨酸残基处仅有一个磷酸化。

【背景】生物学背景:蛋白质 - ...

In vitro AMPylation Assays Using Purified, Recombinant Proteins
Author:
Date:
2017-07-20
[Abstract]  Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation ... [摘要]  翻译后蛋白质修饰(PTM)协调各种蛋白质的活性并确保其功能正常。虽然诸如磷酸化或糖基化的修饰被很好地理解,但是更不寻常的修饰,包括亚硝基化或AMP化仍然比较差的表征。关于蛋白质AMP化的研究 - 其是将AMP部分共价加成到丝氨酸,苏氨酸或酪氨酸的侧链,已经经历了复兴(Yarbrough et al。,2009; Engel et al。 2012年; Ham等人,2014年; Woolery等人,2014年; Preissler等人,2015年; ; Sanyal等人,2015; Truttmann等人,2016; Truttmann等人,2017)。鉴定和表征含丝状(fic)结构域的AMPylases引起了对该PTM的新兴趣(Kinch等人,2009; Yarbrough等人,2009)。基于最近的体内和体外研究,我们现在知道分泌的细菌AMPylase共价连接AMP到Rho家族GTP酶的成员,而后生动物AMPylases修饰HSP70家族蛋白在细胞质和内质网(ER)(Itzen等人,2011; Hedberg和Itzen,2015; Truttmann和Ploegh,2017)。认为AMP化学将HSP70置于不能参与蛋白质重折叠反应的引发剂但瞬时失活的状态(Preissler等人,2015)。 ...

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