Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling

Sameer Ahmed Bhat; Rachel Ferguson; Ritika Adhikari; Shafi Kuchay

doi:10.1016/j.xpro.2023.102416

Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling

STAR Protoc. 2023 Sep 15;4(3):102416. doi: 10.1016/j.xpro.2023.102416. Epub 2023 Jul 4.

Authors

Sameer Ahmed Bhat¹, Rachel Ferguson¹, Ritika Adhikari¹, Shafi Kuchay²

Affiliations

¹ Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, MBRB #1252, Chicago, IL 60607, USA.
² Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, MBRB #1252, Chicago, IL 60607, USA. Electronic address: kuchay@uic.edu.

Abstract

Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine. For complete details of this protocol, please refer to Liang et al.¹.

Keywords: Cell Membrane; Cell-based Assays; Protein Expression and Purification.

MeSH terms

Fluorocarbon Polymers*
Lipid Metabolism
Membrane Lipids*
Polyvinyls / metabolism
Proteins* / metabolism

Substances

polyvinylidene fluoride
Membrane Lipids
Proteins
Polyvinyls
Fluorocarbon Polymers

Grants and funding

R35 GM137452/GM/NIGMS NIH HHS/United States