Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation

Claudia Schirra; Nadia Alawar; Ute Becherer; Hsin-Fang Chang

doi:10.1007/978-1-0716-3135-5_11

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation

Methods Mol Biol. 2023:2654:159-167. doi: 10.1007/978-1-0716-3135-5_11.

Authors

Claudia Schirra¹, Nadia Alawar¹, Ute Becherer¹, Hsin-Fang Chang²

Affiliations

¹ Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany.
² Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany. [email protected].

PMID: 37106182
DOI: 10.1007/978-1-0716-3135-5_11

Abstract

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

Keywords: Immunoisolation; Lytic granule; Magnetic beads; Mass spectrometry; Nitrogen cavitation; Subcellular fractionation; Sucrose density gradient centrifugation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Fractionation / methods
Centrifugation, Density Gradient / methods
Cytological Techniques
Cytoplasmic Granules
Mice
Organelles
Proteins* / metabolism
Subcellular Fractions / metabolism
Vesicle-Associated Membrane Protein 2* / analysis
Vesicle-Associated Membrane Protein 2* / metabolism

Substances

Vesicle-Associated Membrane Protein 2
Proteins