Jun 15, 2026
Expression and Purification of ATG2A
- Elisabeth Holzer1,
- Justyna Sawa-Makarska1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Elisabeth Holzer, Justyna Sawa-Makarska 2026. Expression and Purification of ATG2A. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6zdng5b/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: 223983
Keywords: ASAPCRN, purification of atg2a, atg2a, purification, description of expression
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol details the description of Expression and Purification of ATG2A.
Materials
Buffers and Materials
Lysis Buffer:
| A | B | |
| HEPES (pH 8.0) | 50 mM | |
| NaCl | 300 mM | |
| glycerol | 10% | |
| TCEP | 1 mM | |
| urea | 0.5 M | |
| n-octyl-β-D-glucopyranoside | 0.2% | |
| cOmpleteTM EDTA-free protease inhibitor cocktail | 1× | |
| Benzonase | 1 µL/50 mL | |
| Protease Inhibitor Cocktail | 200 µL/50 mL |
- 0.2% n-octyl-β-D-glucopyranosideGlyconCatalog #D97001-10g
- cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
- Benzonase® NucleaseMerck Millipore (EMD Millipore)Catalog #E1014-25KU
- Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P8849-5ML
Wash Buffer with Glycerol:
| A | B | |
| HEPES (pH 8.0) | 50 mM | |
| NaCl | 300 mM | |
| Glycerol | 10% | |
| TCEP | 1 mM |
Wash Buffer without Glycerol:
| A | B | |
| HEPES (pH 8.0) | 50 mM | |
| NaCl | 300 mM | |
| TCEP | 1 mM |
Additional Materials:
- FreeStyle™ 293 Expression MediumThermo Fisher ScientificCatalog #12338026
- Opti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
- Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K™)Polysciences, Inc.Catalog #23966-1
- EX-CELL® 293 Serum-Free Medium for HEK 293 CellsMerck MilliporeSigma (Sigma-Aldrich)Catalog #14571C
- ANTI-FLAG® M2 Affinity GelMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2220
- TEV protease
- Dounce homogenizer
- Centrifuge (e.g., HITACHI model)
- Liquid nitrogen
Protocol materials
Benzonase® NucleaseMerck Millipore (EMD Millipore)Catalog #E1014-25KU
Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P8849-5ML
Opti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
0.2% n-octyl-β-D-glucopyranosideGlyconCatalog #D97001-10g
EX-CELL® 293 Serum-Free Medium for HEK 293 CellsMerck MilliporeSigma (Sigma-Aldrich)Catalog #14571C
ANTI-FLAG® M2 Affinity GelMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2220
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
FreeStyle™ 293 Expression MediumThermo Fisher ScientificCatalog #12338026
Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K™)Polysciences, Inc.Catalog #23966-1
FLAG PeptideMerck MilliporeSigma (Sigma-Aldrich)Catalog #F3290-4MG
Protein Expression
4d 0h 35m
Culture FreeStyle™ HEK293F cells at 37 °C in FreeStyle™ 293 Expression Medium.
The day before transfection, seed cells at a density of 0.7 × 10⁶ cells/mL.
On the day of transfection, 400 µg of endotoxin-free plasmid DNA encoding 3XFLAG-TEV-ATG2A or its variants was diluted in 13 mL Opti-MEM™ medium
In parallel, 800 µg PEI 25K was also diluted in 13 mL Opti-MEM™.
Combine DNA and PEI mixtures and incubate for 00:15:00 at Room temperature , then add dropwise to a 400 mL culture of HEK293F cells.
15m
24 h post-transfection, culture the supplement with 100 mL EX-CELL® 293 Serum-Free Medium.
1d
72 h later, harvest cells by centrifugation at 270 x g for 00:20:00 .
3d 0h 20m
Wash Pellets once with PBS, flash-frozen in liquid nitrogen, and store at -80 °C .
Cell Lysis
1h
Thaw Frozen pellets On ice and resuspend in 20 mL lysis buffer.
Resuspension is performed by gentle pipetting, followed by mechanical lysis using a Dounce homogenizer:
- 10 strokes with loose pestle
- 2 × 20 strokes with tight pestle
Incubate Lysates for 20-30 min On ice .
30m
Clarify lysates by centrifugation at 15000 rpm for 00:30:00 at 4 °C .
30m
Collect the supernatant and proceed to purification.
FLAG-Affinity Purification
3h
Incubate cleared lysates with 0.6 mL pre-equilibrated anti-FLAG® M2 resin for 03:00:00 at 4 °C with gentle rotation.
3h
Wash beads three times with 13 mL wash buffer containing glycerol.
Wash beads three more times with 13 mL wash buffer without glycerol.
TEV Protease Elution
Transfer washed beads to 1.5 mL tubes.
Add 200 µL of wash buffer without glycerol containing 5 µL TEV protease per tube.
Note
Alternatively, the protein can be eluted using 100 µL of FLAG peptide FLAG PeptideMerck MilliporeSigma (Sigma-Aldrich)Catalog #F3290-4MG at a concentration of 4 mg/mL .
Incubate overnight at 4 °C with gentle agitation.
Collect eluate (Elution 1).
Wash beads once with 150 µL of wash buffer without glycerol to recover residual protein (Elution 2).
Aliquot Elution 1 and Elution 2, snap-freeze in liquid nitrogen, and store at Temperature -80 °C.
Note
All steps were performed at 4 °C or On ice to maintain protein stability. OG was used from a 10% stock in water (CMC ≈ 0.6%) to aid solubilization of membrane- associated ATG2A.