Alexa fluor 680 fluorophore

Apo-SOD1 (10 µM) was incubated with 1 mM alkynyl lipids (a-HNE, a-Ch, a-Seco A and a-Seco B) in 50 mM phosphate buffer pH 8.4 containing 150 mM NaCl and 100 µM DTPA for 24 h at 37 °C. The samples were then reduced with 5 mM sodium borohydride for 1 h at room temperature to stabilize possible adducts, and finally neutralized with 10% HCl. The following click reagents were added to each of the samples: azido-biotin reagent (0.2 mM), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand (0.2 mM), copper sulfate (1 mM), and sodium ascorbate (1 mM), and the samples were vortexed and allowed to stir for 2 h in the dark at room temperature. Biotinylated samples were resolved by SDS-PAGE as described above. The protein was transferred electrophoretically to a polyvinylidene fluoride membrane (Life Technologies, Grand Island, NY) and probed with streptavidin conjugated with the Alexa Fluor 680 fluorophore (Life Technologies). Biotinylated proteins were visualized using the Odyssey Infrared Imaging System and Odyssey software according to the manufacturer (Licor, Lincoln, NE).