Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.

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 substrates in vitro.