Protocol for the purification of replisomes from the Xenopus laevis egg extract system for single-particle cryo-EM analysis

Abstract

Summary: Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM).For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Model Organisms
• Protein expression and purification
• Cryo-EM