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

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.