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Date:
2021-01-05
[Abstract] Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore assembly during mitosis. Here, we describe an in vitro CDK1 kinase assay to detect CENP-C phosphorylation using Phos-tag SDS-PAGE without radiolabeled ATP. Our protocol has advantages in ease and safety over conventional phosphorylation assays using [γ-32P]-ATP, which has potential hazards despite their better sensitivity. The protocol described here can be applicable to other kinases and be also useful for analysis of phospho-sites in ...
[摘要] [摘要]在有丝分裂过程中,准确的染色体分离需要动线粒体(一种大型蛋白复合物),使染色体与纺锤体微管之间形成联系。必需的线粒体成分CENP-C被细胞周期蛋白B-细胞周期蛋白依赖性激酶1(CDK1)磷酸化,该激酶是有丝分裂进程的主要激酶,在有丝分裂过程中促进适当的线粒体组装。在这里,我们描述了一种体外CDK1激酶测定法,可使用Phos -tag SDS-PAGE检测CENP-C磷酸化而无放射性ATP 。ø乌尔协议具有使用[γ-在容易且安全优于常规磷酸化试验的优点32 P] -ATP ,其具有潜在危险,尽管其敏感性更高。该协议describ编这里可以适用于其他激酶并且也用于在基板磷酸位点的分析是有用的体外。
[背景]细胞周期蛋白-B细胞周期蛋白依赖性激酶1(CDK1)是有丝分裂的主要调节剂,其磷酸化许多靶标以确保有丝分裂的进展(Nurse,1990 ; Malumbres and Barbacid ,2005 )。在有丝分裂期间,携带遗传信息的染色体被平均分为两个子细胞。线粒体是关键的大蛋白复合物,通过在染色体和纺锤体微管之间架桥来确保忠实的染色体分离(Fukagawa和Earnshaw,2014)。组成动线粒的各种蛋白质被CDK1磷酸化(Gascoigne等人,2013; Nishino等人,2013; Hara等人,2018b; ...
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