Jun 16, 2026
Conjugation of GABARAP to ATG9A Compartments
- Elisabeth Holzer1,
- Justyna Sawa-Makarska1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Elisabeth Holzer, Justyna Sawa-Makarska 2026. Conjugation of GABARAP to ATG9A Compartments. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodbzzg4o/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
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Created: July 31, 2025
Last Modified: June 16, 2026
Protocol Integer ID: 223988
Keywords: ASAPCRN, conjugation of gabarap, atg9a compartments this protocol, atg9a compartment, gabarap, conjugation
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes the conjugation of GABARAP to ATG9A Compartments.
Materials
- ChromoTek GFP-Trap® AgaroseProteintechCatalog # gta-20
Reaction Buffer:
| A | B | |
| HEPES, pH 7.5 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Lipidation Reaction Mix:
| A | B | |
| ATP | 2 mM | |
| MgCl₂ | 1 mM | |
| Human ATG12–ATG5–ATG16L1 complex | 250 nM | |
| Mouse ATG7 | 250 nM | |
| Human ATG3 | 250 nM | |
| GABARAP | 500 nM |
Preparation of Beads with ATG9A Vesicles or MOCK Controls
Isolate ATG9A-containing compartments or MOCK preparations (from HAP1 wild-type cells).
Incubate each with 10 µL GFP-Trap agarose beads (ChromoTek, Cat# GTA-20).
Mix on a rotating wheel Overnight at 4 °C .
Bead Washing
Wash beads twice with 200 µL Reaction Buffer:
| A | B | |
| HEPES, pH 7.5 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Lipidation Reaction Setup (20 µL total volume)
Prepare a reaction mix with the following final concentrations:
| A | B | |
| ATP | 2 mM | |
| MgCl₂ | 1 mM | |
| Human ATG12–ATG5–ATG16L1 complex | 250 nM | |
| Mouse ATG7 | 250 nM | |
| Human ATG3 | 250 nM | |
| GABARAP | 500 nM |
Note
Note on GABARAP:
- Use GABARAPΔL117 mutant (C-terminal leucine removed) for lipidation.
- Include full-length GABARAP as a negative control (cannot be lipidated).
- Include synthetic liposomes as positive control:
o Lipid composition:
20.2% DOPC, 1.7% DOPS, 43.2% DOPE, 33.6% cholesterol and 1.3% sphingomyelin
Incubation
3h
Incubate reactions at 30 °C for 03:00:00 .
3h
Post-reaction Processing
Wash beads once with 200 µL Reaction Buffer.
Resuspend beads in SDS loading buffer.
Analyze proteins by SDS-PAGE using a 15% gel.
Detection
Detect GABARAP and ATG9A by Western blotting.