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Company: Sigma-Aldrich
Catalog#: 43815
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MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
[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
[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)。 ...

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria
[Abstract]  The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we describe a new protocol that is capable of identifying only cell surface exposed proteins that play a role in immunity, providing this research field with a more straightforward approach to identify novel vaccine targets. Even though N. meningitidis is used as a model in the protocol herein described, this protocol can be used for any Gram-negative bacteria provided modifications and optimizations are carried out to ... [摘要]  脑膜炎球菌(脑膜炎奈瑟氏球菌)仍然是全球人类健康的重大威胁。这种革兰氏阴性细菌导致感染个体的残疾和死亡率升高。尽管有几种可用的疫苗,目前还没有针对所有循环脑膜炎球菌菌株的通用疫苗(Vogel等人,2013)。在这里,我们描述了一种能够识别仅在细胞表面暴露的蛋白质在免疫中发挥作用的新方案,为该研究领域提供了一种更直接的方法来鉴定新的疫苗靶标。即使使用脑膜炎奈瑟氏球菌作为本文所述方案中的模型,该方案可用于任何革兰氏阴性细菌,提供修饰和优化以使其适应不同的细菌和疾病特征(例如薄膜脆性,生长方法,血清抗体水平,等等)。

背景 尝试开发针对N型的新型疫苗。脑膜炎脑膜炎常常依赖于2D SDS-PAGE(二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳)和蛋白质印迹,随后MS(质谱)(Wheeler等人,2007))。然而,这种方法采用全细胞裂解物,鉴定出不具有疫苗潜力的大量蛋白质(Mendum等人,2009)。因此,我们旨在开发一种能够鉴别可能在免疫中起重要作用的细胞表面暴露蛋白质的方法。简言之,我们的方案包括生长感兴趣的病原体,用免疫个体的血清免疫沉淀表面抗原,并通过液相色谱 - ...