Assay for Peroxisomal β-Oxidation Activity

Fibroblasts were homogenized in 0.25 M STKM buffer (0.25 M sucrose, 25 mM HEPES-KOH, pH 7.4, 25 mM KOAc, 5 mM MgCl₂, 0.1 mM EDTA, 1× Roche Complete Protease Inhibitor, 1 mM DTT) and sonicated for 5 min using a BioRuptor (Diagenode) at low power. Lysates were centrifuged at 1,000 × g for 10 min at 4°C, and the resultant supernatant was centrifuged at 20,000 × g for 20 min at 4°C to yield a pellet enriched for peroxisomes. The protein content of peroxisome-enriched subcellular fractions was determined using a Qubit II fluorimeter. Peroxisomal ß-oxidation enzymatic activity of subcellular fractions was measured essentially as described (Lazarow and de Duve, 1976).