Protocol to identify E3 ligases amenable to biodegraders using a cell-based screening

Abstract

Summary: Here, we provide a protocol for the identification of E3 ubiquitin ligases that are functional when implemented as biodegraders using a cell-based screening assay. We describe steps for establishing a stable cell line expressing a GFP-tagged protein of interest (POI), preparing a sub-library of E3 ligases to screen, and performing the cell-based screening. This protocol can be broadly applied to identify any functional E3 ligase in a biodegrader setting.For complete details on the use and execution of this protocol, please refer to Cornebois et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• cell biology
• cell culture
• cell-based assays
• molecular biology
• antibody