Purification of autophagosomes from rat hepatocytes.
To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.
Seglen
Per O
PO
Proteomics Section DNR, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway. Per.O.Seglen@rr-research.no
Brinchmann
Monica Fengsrud
MF
D000818:Animals; D001343:Autophagy; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar
D008869:Microtubule-Associated Proteins; C526109:Anxa3 protein, rat