Immunoblot analysis of MCAD protein.
To evaluate the quantity of MCAD protein in mouse tissue, liver samples from 6–8-wk-old MCAD+/+ (n = 1) and MCAD−/− (n = 3) mice were immediately frozen in liquid nitrogen and stored at −80 °C. For analysis, tissue was homogenized and lysed in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1% SDS) with 10% glycerol and Complete Protease Inhibitor, (Roche Diagnostics Corporation, Indianapolis, Indiana, United States), 1 mM of phenylmethylsulfonylfluoride, and 1 mM of sodium orthovanadate. Lysates were quantified by Bradford BCA protein assay (Bio-Rad, Hercules, California, United States). Protein lysates were denatured, separated in 8% SDS-PAGE, and transferred overnight onto a 0.45 μm nitrocellulose membrane (Schleicher and Schuell, Keene, New Hampshire, United States). After blocking with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween-20, the membrane was immunoblotted overnight at 4 °C with 1:500 dilution of an anti-MCAD antibody. Blots were incubated in anti-mouse IgG HRP-conjugated secondary antibody for 2–4 h at room temperature using standard procedures and developed by chemiluminiscence (Renaissance, NEN Lifesciences Products, Boston, Massachusetts, United States).