| 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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| Ribosome Fractionation in Yeast
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Author:
Date:
2012-08-20
[Abstract] This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the ...
[摘要] 该协议描述了在蔗糖梯度中的酵母核糖体分级。 在翻译的循环过程中，小（40S）和大（60S）核糖体亚基与mRNA结合以形成80S复合物（单体）。 这种核糖体在翻译延长过程中沿着mRNA移动，然后在终止时解离成40S和60S亚基。 在由一个核糖体延伸期间，另外的核糖体可以在相同的mRNA上启动翻译以形成多核糖体。 每个多核糖体复合物的质量主要由其包含的核糖体的数量决定。 因此，通过蔗糖密度梯度离心，基于mRNA上核糖体的加载，可以对细胞内多核糖体群进行大小分级。 几种化合物有助于维持或破坏多核糖体（图1）。
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