Isolation of lysosomes using the Tagless LysoIP method in PBMCs v1

Molecular homeostasis in cells is regulated in part by protein degradation, which is facilitated by the proteasome and lysosomal proteolysis. Lysosomes are membrane bound organelles involved in the turnover of proteins, metabolites and lipids. Recent literature implicates lysosomal dysfunction to be a feature of many a disease, including neurodegenerative diseases. Focused investigation of lysosomal content (proteome/lipidome/metabolome) in disease states could lead to the discovery of novel therapeutics and disease mechanisms. Here we describe our method to isolate peripheral blood mononuclear cells (PBMCs) and perform rapid isolation of intact, tag-less lysosomes from PBMC homogenates using ball-bearing cell breakers and anti-TMEM192 -antibody coupled to magnetic beads. First, cells are broken with physical sheering force as the cell suspension passes through a narrow gap within the cell-breaker, leading to plasma membrane rupture but due to their small size, lysosomes remain intact (Figure 1). Then, the cell homogenate is incubated with the antibody-coupled magnetic beads to allow for rapid immunopurification of lysosomes by binding to the transmembrane protein of lysosomes TMEM192 (Figure 3). The purified lysosomes can be processed and analysed with a variety of techniques including immunoblotting analysis, proteomic/lipidomic/metabolomic tools, fluorescence-activated cell sorting and GCase activity assay. The immunopurification protocol is very fast, less than 15 minutes from start of the incubation with the beads to washed, pure lysosomes. The same protocol can also be used to immunopurify lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293 and A549 cells.