Characterization of acrolein modification of apolipoprotein E3 (apoE3). (A) SDS-PAGE of acrolein modified apoE3. About 10 µg of apoE3 was treated with PBS or increasing amounts of acrolein, electrophoresed on a 4–20% acrylamide gradient gel and stained with Amido Black. The lane assignments for the various acrolein:apoE3 molar ratios are as follows: lane (1) standard, lane (2) unmodified apoE3, lane (3) 1:1, lane (4) 10:1, lane (5) 20:1, lane (6) 40:1, lane (7) 60:1, lane (8) 80:1, lane (9) 100:1. Arrows draw attention to 72 and 130 kDa bands. (B) Western blot of unmodified and acro-apoE3. About 0.5 µg unmodified or acro-apoE3 was electrophoresed and subjected to Western blot using anti-apoE-HRP (left panel) and 5F6 (right panel) antibody. A molar ratio of 10:1 acrolein:apoE3 was used for Western blot. (C) Far-UV CD spectra of unmodified apoE3 and acro-apoE3. Far-UV CD spectra of about 0.2 mg/mL protein were recorded in 10 mM ammonium bicarbonate buffer at pH 7.4, under reduced conditions, in the presence of Tris-(2-carboxyethyl) phosphine (TCEP).