Affinity Purification of Soluble and Membrane-Bound Protein Complexes by a FlpIn Strategy

Rebecca Peschel; Nadine Schmidt; Wolfgang Schliebs; Ralf Erdmann

doi:10.1007/978-1-0716-3048-8_26

Affinity Purification of Soluble and Membrane-Bound Protein Complexes by a FlpIn Strategy

Methods Mol Biol. 2023:2643:373-382. doi: 10.1007/978-1-0716-3048-8_26.

Authors

Rebecca Peschel¹, Nadine Schmidt¹, Wolfgang Schliebs¹, Ralf Erdmann^{1

2}

Affiliations

¹ Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum, Germany.
² Institut für Biochemie und Pathobiochemie, Ruhr-Universität Bochum, Bochum, Germany.

PMID: 36952199
DOI: 10.1007/978-1-0716-3048-8_26

Abstract

For a long time, the isolation of native protein complexes from human cells was accomplished by immunoprecipitation experiments. However, success depends on the quality of the antibodies and the method consumes valuable antibodies, which can hinder subsequent analysis of the isolated complexes. Here, we demonstrate an alternative approach based on affinity purification. It utilizes human Flp-In^TM cells, which genomically express a Protein A-tagged version of the human peroxisomal import receptor PEX5L. Native soluble and membrane-bound complexes containing PEX5L can thereby be isolated via a well-known affinity-based strategy.

Keywords: FlpIn strategy; Human cells; PEX5; Peroxisome; Protein complexes.

MeSH terms

Carrier Proteins* / metabolism
Humans
Peroxisomes* / metabolism
Protein Transport

Substances

Carrier Proteins