Methods for Imaging and Detecting Modification of Proteins by Reactive Lipid Species

Overview

Journal Free Radic Biol Med

Specialties Biochemistry
Biology
General Medicine

Date 2009 May 19

PMID 19446632

Citations 22

Authors

Ashlee N Higdon

Brian P Dranka

Bradford G Hill

Joo-Yeun Oh

Michelle S Johnson

Aimee Landar

Victor M Darley-Usmar

Affiliations

Soon will be listed here.

Abstract

Products of lipid peroxidation are generated in a wide range of pathologies associated with oxidative stress and inflammation. Many oxidized lipids contain reactive functional groups that can modify proteins, change their structure and function, and affect cell signaling. However, intracellular localization and protein adducts of reactive lipids have been difficult to detect, and the methods of detection rely largely on antibodies raised against specific lipid-protein adducts. As an alternative approach to monitoring oxidized lipids in cultured cells, we have tagged the lipid peroxidation substrate arachidonic acid and an electrophilic lipid, 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2), with either biotin or the fluorophore BODIPY. Tagged arachidonic acid can be used in combination with conditions of oxidant stress or inflammation to assess the subcellular localization and protein modification by oxidized lipids generated in situ. Furthermore, we show that reactive lipid oxidation products such as 15d-PGJ2 can also be labeled and used in fluorescence and Western blotting applications. This article describes the synthesis, purification, and selected application of these tagged lipids in vitro.

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