Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation.
ACAD11
Density gradient centrifugation
Liver
Organelle purification
Peroxisomes
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
23
3
2023
pubmed:
24
3
2023
medline:
28
3
2023
Statut:
ppublish
Résumé
Sophisticated organelle fractionation strategies were the workhorse of early peroxisome research and led to the characterization of the principal functions of the organelle. However, even in the era of molecular biology and "omics" technologies, they are still of importance to unravel peroxisome-specific proteomes, confirm the localization of still uncharacterized proteins, analyze peroxisome metabolism or lipid composition, or study their protein import mechanism. To isolate and analyze peroxisomes for these purposes, density gradient centrifugation still represents a highly reliable and reproducible technique. This article describes two protocols to purify peroxisomes from either liver tissue or the HepG2 hepatoma cell line. The protocol for liver enables purification of peroxisome fractions with high purity (95%) and is therefore suitable to study low-abundant peroxisomal proteins or analyze their lipid composition, for example. The protocol presented for HepG2 cells is not suitable to gain highly pure peroxisomal fractions but is intended to be used for gradient profiling experiments and allows easier manipulation of the peroxisomal compartment, e.g., by gene knockdown or protein overexpression for functional studies. Both purification methods therefore represent complementary tools to be used to analyze different aspects of peroxisome physiology. Please note that this is an updated version of a protocol, which has been published in a former volume of Methods in Molecular Biology.
Identifiants
pubmed: 36952174
doi: 10.1007/978-1-0716-3048-8_1
doi:
Substances chimiques
Lipids
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1-12Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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