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

Paolo Passaretti; Milos A Cvetkovic; Alessandro Costa; Agnieszka Gambus

doi:10.1016/j.xpro.2024.103237

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

Paolo Passaretti, Milos A Cvetkovic, Alessandro Costa, Agnieszka Gambus^*

^*Corresponding author for this work

Cancer and Genomic Sciences

Research output: Contribution to journal › Article › peer-review

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Abstract

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

Original language	English
Article number	103237
Number of pages	28
Journal	STAR Protocols
Volume	5
Issue number	3
Early online date	8 Aug 2024
DOIs	https://doi.org/10.1016/j.xpro.2024.103237
Publication status	Published - 20 Sept 2024

Bibliographical note

Keywords

Cryo-EM
Model Organisms
Protein expression and purification

ASJC Scopus subject areas

General Neuroscience
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

Access to Document

10.1016/j.xpro.2024.103237Licence: Creative Commons: Attribution (CC BY)

PassarettiP2024ProtocolFinal published version, 7.57 MBLicence: Creative Commons: Attribution (CC BY)

Cryo-EM studies of a metazoan replisome captured ex vivo during elongation and termination
Passaretti, P. (Co-Investigator) & Gambus, A. (Principal Investigator)
Biotechnology & Biological Sciences Research Council
1/03/24 → 28/02/27
Project: Research Councils
Replisome unloading: mechanism and importance for cell biology
Gambus, A. (Principal Investigator)
The Wellcome Trust
1/09/19 → 29/08/25
Project: Research

Cite this

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

abstract = "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",

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author = "Paolo Passaretti and Cvetkovic, {Milos A} and Alessandro Costa and Agnieszka Gambus",

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AU - Costa, Alessandro

AU - Gambus, Agnieszka

PY - 2024/9/20

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KW - Model Organisms

KW - Protein expression and purification

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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Projects

Cryo-EM studies of a metazoan replisome captured ex vivo during elongation and termination

Replisome unloading: mechanism and importance for cell biology

Cite this