| Sample Preparation of Telomerase Subunits for Crystallization
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Author:
Date:
2015-08-20
[Abstract] Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of telomerase inhibitors for cancer therapy. We have successfully developed a protocol for large-scale preparation of the TRBD-CR4/5 complex of the medaka telomerase in vitro, and used this method to study the high-resolution structure of the TRBD-CR4/5 complex by X-ray crystallography. This procedure may be also adapted to purify other protein-RNA complexes for structural studies.
[摘要] 端粒酶是在大多数真核生物中复制线性染色体末端的大核糖核蛋白复合物。 体外大规模制备端粒酶核心组分长期以来一直是该领域的一个大挑战，阻碍了对端粒酶的催化机理的理解，以及减慢端粒酶抑制剂对癌症的发展 治疗。 我们成功地开发了用于大规模制备体外的medaka端粒酶的TRBD-CR4/5复合物的方案，并且使用该方法研究TRBD-CR4/5复合物的高分辨率结构， 5复合物通过X射线晶体学。 该程序也可以适于纯化其它蛋白质-RNA复合物用于结构研究。
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| Expression and Purification of the Arabidopsis E4 SUMO Ligases PIAL1 and PIAL2
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Author:
Date:
2015-08-05
[Abstract] The proteins PIAL1 (At1g08910) and PIAL2 (At5g41580) are members of the recently discovered group of plant E4 SUMO ligases. This protocol allows quick and simple expression of the recombinant proteins in Escherichia coli (E. coli) and subsequent affinity purification using a maltose binding protein (MBP) tag. The proteins can be used in SUMOylation reactions, where the MBP part of the protein can be detected with a commercially available antibody, or additional purification steps can be applied.
[摘要] 蛋白质PIAL1（At1g08910）和PIAL2（At5g41580）是最近发现的植物E4 SUMO连接酶的成员。 该方案允许在大肠杆菌（大肠杆菌）中快速和简单地表达重组蛋白，并随后使用麦芽糖结合蛋白（MBP）标签进行亲和纯化。 蛋白质可用于SUMO化反应中，其中蛋白质的MBP部分可用市售的抗体检测，或可应用额外的纯化步骤。
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| Generation of Polyclonal Specific Antibodies
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Author:
Date:
2013-06-05
[Abstract] Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).
[摘要] 对感兴趣的蛋白质特异性的抗体的产生是许多学科中的常见方法。 该方案详述了使用细菌表达的融合蛋白作为抗原在兔中产生多克隆抗体的步骤。 该协议基于Wirschell等人（2013）中提供的数据生成。
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