Protocol for detecting cytosol-retained proopiomelanocortin by fractionation and co-immunoprecipitation assay

Signal peptide (SP)-uncleaved proopiomelanocortin (POMC) accumulates in the cytosol when ER translocation or protein quality control is perturbed. Here, we present a protocol to prepare and validate a POMC SP-specific antibody. We then use this antibody to detect cytosol-retained SP-uncleaved POMC in mammalian cells. We describe steps for using sequential digitonin-based semi-permeabilization followed by Triton X-100 lysis to isolate cytosolic and organelle fractions. We then detect cytosolic POMC by immunoblotting upon genetic depletion or enrich it by SP-specific immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Mun et al.¹.