Methods for imaging and detecting modification of proteins by reactive lipid species.
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.
Higdon
Ashlee N
AN
Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0022, USA.
Dranka
Brian P
BP
Hill
Bradford G
BG
Oh
Joo-Yeun
JY
Johnson
Michelle S
MS
Landar
Aimee
A
Darley-Usmar
Victor M
VM
D016718:Arachidonic Acid / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D001896:Boron Compounds / Q000378:metabolism; D003952:Diagnostic Imaging / Q000295:instrumentation; D005456:Fluorescent Dyes; D007249:Inflammation; D008055:Lipids / Q000138:chemical synthesis / Q000302:isolation & purification; D018384:Oxidative Stress; D019047:Phantoms, Imaging; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011506:Proteins / Q000032:analysis*
C097240:15-deoxy-delta(12,14)-prostaglandin J2; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008055:Lipids; D011506:Proteins; D016718:Arachidonic Acid; D015230:Prostaglandin D2