Jun 15, 2026

Isolation of Autophagosomes and Proteinase K Protection Assay

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6z1q1gqe/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6z1q1gqe/v1
Protocol Citation: Elisabeth Holzer 2026. Isolation of Autophagosomes and Proteinase K Protection Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6z1q1gqe/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
-
Created: August 01, 2025
Last Modified: June 15, 2026
Protocol  Integer ID: 223996
Keywords: ASAPCRN, isolation of autophagosome, autophagosome
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes the isolation of autophagosomes and proteinase K protection assay.
Materials
Vesicle Isolation Buffer (VIB):

AB
HEPES pH 7.520 mM
NaCl150 mM
Sucrose250 mM
cOmplete‱ EDTA-free protease inhibitors (Roche)1×
β-glycerophosphate20 mM
Sodium orthovanadate1 mM
NaF1 mM
EDTA pH 8.01 mM
Wash Buffer (WB):
AB
HEPES pH 7.520 mM
NaCl150 mM
cOmplete EDTA-free protease inhibitors1×
  • Proteinase KThermo Fisher ScientificCatalog #EO0491
  • PMSF SolutionSanta Cruz BiotechnologyCatalog #sc-482875
Equipment
Protein Lobind 1.5mL tubes
NAME
Tubes
TYPE
Eppendorf
BRAND
0030108442
SKU
https://www.eppendorf.com/au-en/eShop-Products/Laboratory-Consumables/Tubes/Protein-LoBind-Tubes-p-0030108116
LINK
1.5mL, snap-cap, Protein LoBind, PCR clean, colourless
SPECIFICATIONS



Isolation of Autophagosomes: Cell Culture and Treatment
2h
Use the following cell lines:

  • HAP1 ATG9A-GFP (RRID:CVCL_E2TR)
  • HAP1 ATG9A-GFP ATG2 DKO (RRID:CVCL_F0LE)
Seed cells in 15 cm dishes (two dishes per condition).
Treat cells with:

  • Earle’s Balanced Salt Solution (EBSS).
  • 100 nanomolar (nM) Bafilomycin A1 (BafA).
Incubate for 02:00:00 prior to harvesting.
2h
Isolation of Autophagosomes: Cell Harvesting and Lysis
Harvest cells by trypsinization and pellet via centrifugation.
Wash once with PBS.
Resuspend pellets in 800 µL Vesicle Isolation Buffer (VIB):

AB
HEPES pH 7.520 mM
NaCl150 mM
Sucrose250 mM
1× cOmplete™ EDTA-free protease inhibitors (Roche)
β-glycerophosphate20 mM
Sodium orthovanadate1 mM
NaF1 mM
EDTA pH 8.01 mM

Transfer to LoBind tubes (Eppendorf, Cat# 0030108116).
Isolation of Autophagosomes: Cell Disruption
10m
Lyse cells by passing suspension through a 26G needle 30×.
Chill lysates On ice for 00:10:00 .
10m
Repeat needle passage another 30×.
Isolation of Autophagosomes: Sequential Centrifugation for Organelle Enrichment
35m
Clear nuclei and debris:
Centrifuge at 1000 x g for 00:10:00 at 4 °C .
10m
Collect supernatant.
Re-extract pellet:

Add 700 µL VIB to the pellet of the previous step, repeat 1000 x g spin.
Pool both supernatants.
Remove residual debris:
Centrifuge pooled supernatants at 2000 x g for 00:05:00 .
5m
Collect supernatant.
Enrich autophagosomes:
Centrifuge supernatant at 10000 x g for 00:20:00 at 4 °C .
20m
Collect pellet.
Washing and Resuspension
20m
Wash pellet with 500 µL Wash Buffer (WB):
AB
HEPES pH 7.520 mM
NaCl150 mM
1× cOmplete EDTA-free protease inhibitors

Re-pellet at 10000 x g for 00:20:00 .
20m
Resuspend final pellet in 500 µL WB + 1 mM DTT
AB
HEPES pH 7.520 mM
NaCl150 mM
DTT1 mM

Use this AP-enriched fraction for downstream applications.
Proteinase K Protection Assay: Protease Treatment
20m
Use 150 µL of AP suspension.
Add 10 µL proteinase K (Thermo Scientific, Cat# EO0491).

Incubate for 00:20:00 at 4 °C .
20m
Proteinase K Protection Assay: Protease Inhibition
10m
Add 1 millimolar (mM) PMSF (Santa Cruz Biotechnology, Cat# sc-482875).
Incubate On ice for 00:10:00 .
10m
Proteinase K Protection Assay: Sample Processing
10m
Centrifuge at 10000 x g for 00:10:00 at 4 °C .
10m
Resuspend pellet in

  • 15 µL WB
AB
HEPES pH 7.520 mM
NaCl150 mM
DTT1 mM
  • 6× SDS loading buffer
Analyze by SDS-PAGE and Western blotting.