Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology
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Author:
Date:
2017-07-20
[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its ...
[摘要] 在肠和呼吸上皮中称为杯状细胞的专门分泌细胞负责粘蛋白的分泌。 粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。 这些大蛋白质被短聚糖链密集取代,其具有许多重要的功能作用,包括测定粘液凝胶的水合和粘弹性质,其保护肠上皮。 在该方案中,我们全面地描述了通过琼脂糖凝胶电泳提取小鼠粘液的方法及其分析。 此外,我们描述了使用高铁二胺 - 阿尔星蓝,周期酸席夫氏 - 阿尔辛蓝和免疫染色方法来鉴别和区分这些粘蛋白糖蛋白上糖基化的不同状态,特别是侧重于硫酸化和唾液酸化。 【背景】一层粘液保护肠上皮,主要由粘蛋白,水,蛋白质和无机盐组成。粘液屏障的粘性和凝胶状特性使其能够物理保护和润滑粘膜,主要由粘蛋白赋予。粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。然而,粘蛋白主要用O-聚糖糖装饰,其占分子量的80%。不同的位点特异性和粘蛋白特异性糖基化模式影响粘蛋白和粘液凝胶的性质。众所周知,粘蛋白糖基化在感染和疾病中发生改变(Arike等人,2017; ...
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Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method
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Author:
Date:
2017-06-20
[Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor. We have shown that Thermofluor can also be applied to predict binding of substrates and inhibitors to a yeast α-glucosidase. The methods described here in detail were used in Viigand et al., 2016.
[摘要] α-葡糖苷酶(包括麦芽糖酶和异麦芽糖酶)是从一组α-葡糖苷底物释放葡萄糖的酶。 可以使用该特征来测定其催化活性,底物特异性和热稳定性。 蛋白质的热稳定性也可以使用高通量差示扫描荧光测定法(也称为Thermofluor)来测定。 我们已经表明,Thermofluor也可以应用于预测底物和抑制剂与酵母α-葡萄糖苷酶的结合。 这里详细描述的方法用于Viigand等人,2016。 【背景】麦芽糖酶(EC 3.2.1.20)和异麦芽糖酶(EC 3.2.1.10)是根据CAZy分类属于糖苷水解酶家族13的α-葡糖苷酶(Lombard等,2014)。甲基营养酵母多形汉酵母的麦芽糖酶MAL1是非选择性的,它将产生D-葡萄糖的麦芽糖和异麦芽糖状α-葡萄糖苷水解为反应产物之一。因此,麦芽糖酶对其底物的活性可以根据葡萄糖释放来确定。该工作描述的葡萄糖液色辅助方法可以快速方便地测定麦芽糖酶的活性,底物特异性和热稳定性。重要的是,这种基于活性的方法可以适用于产生葡萄糖作为反应产物的其它酶。高通量Thermofluor方法主要用于蛋白质晶体学测量(热)稳定性蛋白质(Boivin等,2013; Ericsson等,2006)。我们使用Thermofluor ...
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Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting
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Author:
Date:
2017-05-05
[Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).
[摘要] 腺相关病毒(AAV)是一种小型单链DNA病毒,需要存在辅助病毒,如腺病毒或疱疹病毒,以有效地复制其基因组。 AAV DNA通过滚转发夹机制(Ward,2006)进行复制,并且在复制期间可以检测出几种DNA中间体。该详细方案描述了如何使用改良的Hirt提取物(Hirt,1967)程序和Southern印迹(Southern,1975)分析在AAV复制期间形成的AAV DNA中间体。
背景 AAV DNA复制通过滚动发夹机制在由AAV和辅助病毒如腺病毒或疱疹病毒共感染的细胞中进行(Ward,2006)。 AAV DNA由4.7kb的线性DNA分子和倒置的末端重复(ITR)组成,折叠形成T形发夹结构。 3'末端发夹作为AAV DNA复制的引物。这些发夹结构由AAV Rep蛋白再生,允许进一步复制(Im和Muzyczka,1990)。 AAV DNA的+和 - 链都被包装并且是感染性的(Rose等人,1969)。当分析复制AAV DNA时,可以检测到几种复制中间体(Straus等人,1976)。最丰富的复制中间体是由AAV DNA的一个和一个链形成的线性单体双链体分子,其被认为是将包装在预先形成的衣壳中的后代单链分子的直接前体(Straus ,1976)。二聚体复制中间体也是常见的,AAV复制模型与甚至更大的复制中间体相容。 ...
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