Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
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Author:
Date:
2017-05-05
[Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K+ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)
[摘要] 基于交配的分ubiquitin(mbSUS)测定是具有许多优点的经典酵母双杂交系统的替代方法。 mbSUS测定依赖于泛素降解途径作为蛋白质 - 蛋白质相互作用的传感器,并且它适用于测定细胞溶质或膜结合的全长蛋白质之间的相互作用。在这里,我们描述了已经用于检测K + 通道和SNARE蛋白之间的相互作用的mbSUS测定方案(Grefen等人,2010和2015; Zhang 等等,2015和2016)
背景 图1是mbSUS测定的概况。泛素部分被分成两半,N末端半突变(NubG)以避免重组。泛素部分(Cub)的C末端一半与转录报告基因复合物PLV(Protein A-LexA-VP16)连接。两种蛋白质(X和Y)分别与NubG和CubPLV融合产生蛋白质 - 蛋白质相互作用分析系统。转化后,酵母菌株THY.AP5含有NubG-X融合蛋白,而酵母菌株THY.AP4含有Y-CubPLV融合蛋白。在交配后,在二倍体酵母中,如果蛋白质X和Y彼此相互作用,则将重新组装功能性泛素,这导致PLV的切割。释放的转录蛋白复合物PLV可以开启报告基因(ADE2,HIS3),并允许酵母生长在选择性培养基上。
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Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth
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Author:
Date:
2017-04-05
[Abstract] We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson-Thomas et al., 2015) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method (Nader et al., 1999) which we optimized for archeological samples.
[摘要] 我们开发了从考古学骨骼和牙齿中分离蛋白聚糖和糖胺聚糖的方法。 以前已经公开了这些方法(Coulson-Thomas等人,2015),并且在这里更详细地描述。 在糖胺聚糖的情况下,该方法是先前描述的方法(Nader等人,1999),其针对考古样品进行了优化。 【背景】骨组织主要由矿物成分(羟基磷灰石)和由胶原,非胶原蛋白和蛋白聚糖(PG)组成的有机基质组成。 由于与羟基磷灰石紧密结合,细胞外基质蛋白和PG被保护免受死亡后温度和化学药剂的破坏作用。 然而,到目前为止,只有DNA和蛋白质已经从考古学骨骼中成功提取,因此我们开发了从考古学骨骼和牙齿中分离PG和糖胺聚糖(GAG)链的方法。 PGs和GAG在骨形态发生,体内平衡和退行性骨病中起主要作用,从考古骨骼分析这些分子将揭示有价值的古生物学信息。
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Protein Sample Preparation for Proteomic Analysis in Leishmania donovani
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Author:
Date:
2014-03-05
[Abstract] Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional over pre-transcriptional regulatory mechanisms (Clayton, 2002). Thus proteomic analysis has proven an essential tool for understanding pathways implicated in Leishmania infectivity, host-parasite interactions, drug resistance and others. When employing a comparative proteomics analysis between different parasitic cell lines, it is essential that these lines are cultivated in exactly the same way, in the same cell density and growth phase. More importantly when ...
[摘要] 利什曼原虫属是锥虫病原生动物属,是负责疾病利什曼病的寄生虫。这些原生动物,与其他高等真核生物相比,以非典型方式调节它们的基因表达。基因表达的调节的特征在于转录后优先于转录前调节机制(Clayton,2002)。因此,蛋白质组学分析已经证明是了解利什曼原虫感染,宿主寄生虫相互作用,耐药性和其他相关途径的重要工具。当在不同寄生细胞系之间使用比较蛋白质组学分析时,以相同的方式,在相同的细胞密度和生长期中培养这些细胞系是必要的。更重要的是,当怀疑细胞周期缺陷时,必须在同一细胞周期阶段同步细胞系,以消除可能的人工因素。该方案描述了在杜氏利什曼原虫( donovani )中用于蛋白质组分析的全蛋白样品的制备。
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