Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis

  • Curr Protoc. 2024 May;4(5):e1042. doi: 10.1002/cpz1.1042.
Hang T Huynh  1  2 ,  Evgeniia Shcherbinina  1  2 ,  Hsiang-Chi Huang  1  2 ,  Reyhaneh Rezaei  1  2 ,  Aishe A Sarshad  1  2
Affiliations
  • 1. Department of Medical Biochemistry and Cell biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
  • 2. Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.
PMID: 38767195 DOI: 10.1002/cpz1.1042
Abstract

Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.

Keywords
biochemical fractionation; cellular compartmentalization; cytoplasmic extraction; nuclear isolation; protein localization.