Preparation of Cell Lysates of Fission Yeast for Immunoprecipitation.
Preparation of Cell Lysates of Fission Yeast for Immunoprecipitation.
复制标题
用于免疫沉淀的裂殖酵母细胞裂解物的制备。
DOI：
10.1007/978-1-4939-7546-4_12
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发表时间：
2018
影响因子：
--
通讯作者：
Minoru Yoshida
中科院分区：
文献类型：
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作者：
Akihisa Matsuyama;Atsuko Shirai;Minoru Yoshida
免疫沉淀是检测和纯化目的蛋白质最重要和最广泛使用的技术之一。由于抗原和抗体之间的高度特异性相互作用，目标蛋白被有效地纯化和浓缩。为了获得合理量的靶蛋白，制备其中靶蛋白以可溶形式存在的总细胞裂解物至关重要。在这里，我们描述了制备裂殖酵母总细胞裂解物的方法，然后将其直接用于免疫沉淀。我们还描述了一些选择试剂来制备对蛋白质溶解度有重大影响的缓冲液的技巧，因为基本上没有试剂可以容纳具有不同特性的全部蛋白质。
Immunoprecipitation is one of the most important and widely used techniques for the detection and purification of a protein of interest. Thanks to highly specific interaction between antigen and antibody, a target protein is purified and concentrated effectively. To obtain reasonable amounts of a target protein, it is crucially important to prepare total cell lysates in which the target protein is present in a soluble form. Here, we describe methods to prepare total cell lysates of fission yeast, which are then used directly for immunoprecipitation. We also describe some tips to select reagents for preparing buffers having a substantial impact on protein solubility, because there is essentially no reagent that can accommodate the full range of proteins having different characteristics.