| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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| In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein
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Author:
Date:
2014-11-05
[Abstract] Glycosyltransferases are carbohydrate active enzymes containing catalytic modules involved in catalysing the biosynthesis of glycosidic bonds in oligo- and polysaccharides and glycoconjugates. One of the most comprehensive collections of Carbohydrate Active enZYmes is the CAZy database (http://www.cazy.org) comprising 120,000 glycosyltransferases allocated to 96 families based mainly on sequence homologies of their conserved and catalytically active domains (Cantarel et al., 2009). Interestingly, the glycosyltransferase activities of only about 1.6% of these proteins have been experimentally characterized (Lombard et al., 2014). In recent years, membrane-bound glycosyltransferases of a number of families have been shown ...
[摘要] 糖基转移酶是含有催化模块的碳水化合物活性酶，其涉及催化寡糖和多糖和糖缀合物中糖苷键的生物合成。 Carbohydrate Active enZYmes最全面的集合之一是CAZy数据库（ http://www.cazy.org ） ），其包含分配给96个家族的120,000个糖基转移酶，主要基于其保守和催化活性结构域的序列同源性（Cantarel等人，2009）。有趣的是，仅约1.6％的这些蛋白质的糖基转移酶活性已经通过实验表征（Lombard等人，2014）。近年来，已显示许多家族的膜结合糖基转移酶在植物细胞壁多糖的生物合成中起关键作用（Doblin等人，2010; Scheller和Ulvskov，2010 ; ...
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| Tandem Affinity Purification in Drosophila Heads and Ovaries
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Author:
Date:
2012-08-05
[Abstract] Tandem affinity purification (TAP) (Pugi et al.,2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells(Pugi et al.,2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X ...
[摘要] 串联亲和纯化（TAP）（Pugi等人，2001; Rigaut等人，1999）是使用目标靶蛋白的标记方法的方法两步纯化方案以在天然条件和表达水平下下拉蛋白复合物。 TAP标签由三种组分组成：钙调素结合肽，烟草蚀纹病毒（TEV）蛋白酶切割位点和作为免疫球蛋白G（IgG）结合结构域的蛋白A.该方案从酵母细胞中使用的原始方法（Pugi等人，2001; Rigaut等人，1999）修饰，用于从果蝇头分离蛋白质复合物，以及卵巢表达感兴趣的TAP标记的蛋白质。为了确定果蝇脆弱X蛋白（dFMR1）的体内结合伴侣，我们开发了表达重组形式的具有羧基末端TAP标签的dFMR1的苍蝇的转基因菌株（Tsai和Carstens，2006）。为了确保构建体在野生型水平表达，我们在救援突变不育表型的基因组拯救构建体的上下文中工程化这种形式的标记蛋白质。使用温和条件进行纯化过程以维持天然蛋白质相互作用。对于在果蝇S2细胞培养物中的TAP方法，我们成功地使用了由Tsai和Carstens先前公布的方案（Tsai和Carstens，2006; Bhogal等人，2011）。
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