Work

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

Year: 2026

Type: article

Abstract: 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

Source: STAR Protocols

DOI: 10.1016/j.xpro.2026.104662

Authors: Sang-Hyeon Mun, Cheol-Sang Hwang

Institution: Korea University

Language: English

Topic: Endoplasmic Reticulum Stress and Disease

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550