Autolysosome/lysosome purification was performed with a lysosome isolation kit (LYSISO1, Sigma-Aldrich). The crude autolysosomes/lysosomes were obtained by differential centrifugation. And then the lysosome/autolysosome fraction was further purified from above preparation using optiprep gradient centrifugation. The fraction was collected and resuspended in extraction buffer containing protease inhibitor cocktail. To obtain pure lysosome/autolysosomes, this above fraction, containing autolysosomes/lysosomes, was diluted with 19% Optiprep density gradient medium solution, and overlaid with 16%, 12%, 8%, 5% and 3%. Optiprep solutions. After centrifugation, the fractions were analyzed by western blotting and TEM. The purified autolysosomes/lysosomes were collected and used for the in vitro assays.
The purified lysosomes/autolysosomes were washed twice by PBS. The pellet was suspended by 900 μl of 10 mM NHS-biotin solution and incubated for 30 min. The reaction was stopped by addition of 0.5 M TBS. The mixture were spinned at 20,000 × g at 4 °C, and resuspended with 100 μl of motility assay buffer for AFM assay. The pulldown assay was done by using streptavidin-conjugated beads, and the labeling efficiency was detected by western blotting.
The purified lysosomes/autolysosomes were washed twice by PBS. The pellet was suspended by 900 μl of 10 mM NHS-biotin solution and incubated for 30 min. The reaction was stopped by addition of 0.5 M TBS. The mixture were spinned at 20,000 × g at 4 °C, and resuspended with 100 μl of motility assay buffer for AFM assay. The pulldown assay was done by using streptavidin-conjugated beads, and the labeling efficiency was detected by western blotting.