Protocol to detect OLA1 polyubiquitination by Aurora A in vivo and in vitro

Aurora A is a critical kinase that functions in centrosome maturation and bipolar spindle assembly. On the other hand, Aurora A has E3 ubiquitin ligase activity and polyubiquitinates Breast cancer gene 1 (BRCA1)-interacting protein Obg-like ATPase 1 (OLA1), targeting it for proteasomal degradation. Here, we present a protocol to detect OLA1 ubiquitination. We describe steps for recovering frozen cells and protein purification. We then detail assays for both in vivo and in vitro ubiquitination of OLA1 by Aurora A. For complete details on the use and execution of this protocol, please refer to Fang et al.¹.