The Isolation of Peroxisomes

  • Tsuneo ImanakaEmail author


Peroxisomes are subcellular organelles bound by a single membrane. They are involved in a variety of metabolic processes such as β-oxidation of very long chain fatty acids. Isolated and purified peroxisomes are necessary to obtain a detailed understanding of their structure and function. Here, our three protocols for the isolation of peroxisomes from rat liver, rat hepatoma H4IIE cells and yeast Komagataella phaffii (previously called Pichia pastoris) are presented. Highly pure peroxisomes can be prepared from rat liver by differential centrifugation followed by Nycodenz gradient centrifugation. It is difficult to prepare highly purified peroxisomes from cultured mammalian cells, but the subcellular fractionation of peroxisomes is still a powerful tool to analyze whether a certain protein is localized to peroxisomes. Peroxisomes are potently induced in methylotrophic yeast such as K. phaffii. A large amount of mammalian peroxisomal proteins can be expressed in cells under the control of the alcohol oxidase gene promoter in a medium containing methanol as the only carbon source. Therefore, purified peroxisomes are useful for characterizing mammalian peroxisomal proteins. The protocols for the isolation of peroxisomes by immuno-beads and peroxisomal membranes by a sodium carbonate procedure are also presented.


Peroxisome Peroxisomal membrane Subcellular fractionation Density gradient centrifugation Rat liver Komagataella phaffii 



Endoplasmic reticulum


Peroxisomal membrane protein



The publication is supported in part by a Grant-in-Aid for Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan. Pacific Edit reviewed the manuscript prior to submission.


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© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.Faculty of Pharmaceutical SciencesHiroshima International UniversityKure, HiroshimaJapan
  2. 2.University of ToyamaToyamaJapan

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