In Vivo Epitope Tagging of Plant Mitochondria

Franziska Kuhnert; Andreas P M Weber

doi:10.1007/978-1-0716-1791-5_14

In Vivo Epitope Tagging of Plant Mitochondria

Methods Mol Biol. 2022:2379:253-264. doi: 10.1007/978-1-0716-1791-5_14.

Authors

Franziska Kuhnert¹, Andreas P M Weber²

Affiliations

¹ Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich Heine University, Universitätsstrasse 1, Düsseldorf, Germany.
² Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich Heine University, Universitätsstrasse 1, Düsseldorf, Germany. [email protected].

PMID: 35188666
DOI: 10.1007/978-1-0716-1791-5_14

Abstract

Mitochondria play a key role in cellular metabolism. Analyses of the genome, the proteome, metabolic, physiological, and biochemical functions of mitochondria frequently require the isolation of intact and functional mitochondria from various plant tissues with sufficient yield. For this purpose, we generated a transgenic Arabidopsis thaliana (Arabidopsis) line which presents a triple hemagglutinin tag on the surface of the outer mitochondrial membrane. The affinity tag enables immunocapture of the organelles in a single step. This chapter gives detailed instructions on how to generate transgenic Arabidopsis lines harboring a ubiquitously expressed 3xHA-sGFP-TOM5 mitochondrial fusion protein that is targeted to the outer mitochondrial membrane and enables purification of the organelles in a single step.

Keywords: Arabidopsis; HA-tag; Immunocapture; Magnetic beads; Mitochondria; Organelle isolation; TOM5.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Arabidopsis Proteins* / genetics
Arabidopsis Proteins* / metabolism
Arabidopsis* / metabolism
Epitopes / metabolism
Mitochondria / metabolism
Mitochondrial Membranes / metabolism

Substances

Arabidopsis Proteins
Epitopes