Generation of stable LysoTag expressing cell lines and LysoTag immunoprecipitation of lysosomes 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 how to produce stable LysoTag expressing cell lines and how to perform rapid isolation of lysosomes in cultured cells with immunoprecipitation of the LysoTag using HA-coupled magnetic beads. The immunoprecipitation protocol is very fast, less than 15min from start of the incubation with the beads. The protocol can be used to immunoprecipitate lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293 and A549 cells expressing the LysoTag.