Analyzing Lysosomal Membrane Protein Binding

To analyze GpX4 and GAPDH binding to lysosomal membranes, purified lysosomal fractions (Fraction No. 2) were divided into five parts and resuspended in homogenization buffer (250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 1 mM EDTA) containing 0, 0.1, 0.2, 0.4, or 0.8 M NaCl. The mixtures were incubated for 30 min on ice and subjected to ultracentrifugation at 160,000 ×g for 1 h at 4°C in a fixed angle rotor (TLA120.2; Beckman Coulter). The pellet (lysosomal membrane) was resuspended in 50 μL of SDS-PAGE sample buffer and resolved by SDS-PAGE. The supernatant (protein from the lysosomal membrane) was added to 20% trichloroacetic acid (TCA) to extract protein for 30 min and centrifuged at 20,000 ×g for 10 min at 4°C. The pellet (extract protein) was washed with cold acetone and centrifuged at 20,000 ×g for 5 min at 4°C. The TCA/acetone precipitate was also suspended in 50 μL of SDS-PAGE sample buffer and resolved by SDS-PAGE.³⁰ (link)