Step-by-Step Protocol for Superparamagnetic Nanoparticle-Based Endosome and Lysosome Isolation from Eukaryotic Cell

Methods Mol Biol. 2020:2125:167-172. doi: 10.1007/7651_2019_212.

Abstract

Here, we report our step-by-step protocol for superparamagnetic nanoparticle (SPMNP)-based endosome and lysosome isolation from HeLa. Briefly, we synthesized SPMNP 1.0 with iron oxide (Fe3O4) core using thermal decomposition method. Further, we performed ligand-exchange strategy for surface functionalization of SPMNP 1.0 with dimercaptosuccinic acid (DMSA). Thus, we generated DMSA-SPMNP 2.0 and used DMSA-SPMNP 2.0 to isolate endosomes and lysosome from HeLa cells. Using our SPMNP subcellular fractionation protocol, we are able to isolate high-pure-high-yield lysosomes using DMSA-SPMNP 2.0 for lysosome proteomics and lipidomics in order to better understand subcellular compartments.

Keywords: DMSA; Endosomes; Lysosomes and pulse-chase; Superparamagnetic nanoparticles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Dynamic Light Scattering
  • Endosomes / metabolism*
  • Eukaryotic Cells / metabolism*
  • HeLa Cells
  • Humans
  • Ligands
  • Lysosomes / metabolism*
  • Magnetic Iron Oxide Nanoparticles / chemistry*
  • Magnetic Iron Oxide Nanoparticles / ultrastructure
  • Maleimides / chemistry
  • Nanotechnology / methods*

Substances

  • Ligands
  • Maleimides
  • maleimide