| 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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| Detection of CLEC5A-JEV Interaction by ELISA
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Author:
Date:
2012-10-05
[Abstract] JEV (Japanese encephalitis virus) belonging to Flaiviridae interacts with CLEC5A (C-type lectin domain family 5, member A), a member of C-type lectin associated with DAP12 signaling protein and expressed on myeloid cell, as the same extent as Dengue virus. This protocol is used to perform and determine the interaction between purified JEV particles with human and murine CLEC5A.Fc fusion proteins by ELISA method. The different strain and batch of purified JEV as well as purity of CLEC5A.Fc fusion protein might affect the result of absorbance. Modifications are most likely needed if users intend to perform ELISA assay.
[摘要] 属于黄病毒科的JEV（ 病毒）与CLEC5A（C型凝集素结构域家族5，成员A） C型凝集素，与DAP12信号传导蛋白相关并且在骨髓细胞上表达，与登革热病毒的程度相同。 该方案用于通过ELISA方法进行和确定纯化的JEV颗粒与人和鼠CLEC5A.Fc融合蛋白之间的相互作用。 不同的菌株和批次的纯化JEV以及CLEC5A.Fc融合蛋白的纯度可能影响吸光度的结果。 如果用户打算进行ELISA测定，则很可能需要修饰。
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| Co-immunoprecipitation in Yeast
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Author:
Date:
2012-08-20
[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically binds to that particular protein (antigene protein). The partner proteins that are bound to a specific target protein can be co-immunoprecipitated together with an antigen. These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein. Here I describe the protocols that allow to immunoprecipitate different protein complexes, for example NAC complex (Panasenko et al., 2009), ...
[摘要] 该协议描述了通过共免疫沉淀（CoIP）对面包酵母中蛋白质 - 蛋白质相互作用的研究。 CoIP是鉴定细胞中生理相关的蛋白质 - 蛋白质相互作用的技术。 使用特异性结合该特定蛋白（抗原蛋白）的抗体可以从溶液中分离出感兴趣的蛋白。 与特定靶蛋白结合的伴侣蛋白可以与抗原一起免疫共沉淀。 然后可以分析这些蛋白质复合物以鉴定新的结合配偶体，结合亲和力，结合的动力学和靶蛋白的功能。 这里我描述允许免疫沉淀不同蛋白复合物的方案，例如NAC复合物（Panasenko等人，2009），Ccr4-Not复合物（Panasenko和Collort，2011），核糖体（Panasenko和Collort ，2012），并调查他们的合作伙伴。 对于每个CoIP，我使用不同的裂解缓冲液，如下文配方中所示。
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