Autophagosome immunoisolation from GFP-LC3B mouse tissue

ABSTRACT

We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4–5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The post-nuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis.

Abbreviations: EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LC3: microtubule-associated protein 1 light chain 3 beta; MES: 4-morpholineethanesulfonic acid (MES); SQSTM1: sequestosome 1; TEM: transmission electron microscopy

KEYWORDS:

• Autophagosome
• autophagy
• GFP-LC3
• immunoprecipitation
• isolation

Acknowledgements

We thank Alicia Withrow, PhD of Michigan State University for helping us optimize the negative staining protocol.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported (in part) by a Sybil B. Harrington Physician Scientist Award from Research to Prevent Blindness, New York, NY (DNZ). Additional support was provided by National Eye Institute R01-EY-020823 (DNZ), Foundation Fighting Blindness (DNZ), and University of Michigan Core Center for Vision Research (NEI-EY007003); National Eye Institute (US) [R01-EY-020823]; Research to Prevent Blindness (US); Foundation Fighting Blindness (US).