Jun 15, 2026

Expression and Purification of ATG9A

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmbn6bg3p/v1
  • 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.yxmvmbn6bg3p/v1
Protocol Citation: Julia Romanov, Justyna Sawa-Makarska, Elisabeth Holzer 2026. Expression and Purification of ATG9A. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbn6bg3p/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: 223984
Keywords: ASAPCRN, purification of atg9a, atg9a, 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 ATG9A.
Materials
Buffers and Materials

Lysis Buffer:

AB
HEPES (pH 7.5)50 mM
NaCl500 mM
Glycerol10%
β-mercaptoethanol2 mM
MgCl₂2 mM
1× cOmplete™ EDTA-free protease inhibitor cocktail
Benzonase1 µL/50 mL
Protease Inhibitor Cocktail200 µL/50 mL
  • 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

Solubilization Buffer (same as lysis buffer + detergent):

  • Add 0.3% n-Dodecyl-β-D-maltoside (DDM; Glycon, Cat# D97002-10g)


Elution Buffer:

AB
HEPES (pH 7.5)30 mM
NaCl300 mM
Glycerol10%
β-mercaptoethanol2 mM
DDM0.3%
Variable imidazole80, 150, 200, 300 mM

SEC Buffer:

AB
HEPES (pH 7.5)25 mM
NaCl150 mM
DDT1 mM
DDM0.2%

Additional Materials:

  • 3 Ni-NTA HisTrap HP, 5 x 5 mlCytiva Life SciencesCatalog #17524802
  • Amicon Ultra filter, 100 kDa MWCOMerck MilliporeCatalog #UFC8100
  • Superose 6 Increase 10/300 GL column (Cytiva, Cat# GE17-5172-01)


  • HisTrap HP column (Cytiva, Cat# 17524802)
3 Ni-NTA HisTrap HP, 5 x 5 mlCytiva Life SciencesCatalog #17524802
Protein Expression
15m
The ATG9A construct was designed with an N-terminal 6xHis tag followed by a TEV cleavage site, the coding sequence for ATG9A, GFP, and a Twin-Strep tag. The ATG9A coding sequence was codon-optimized for insect cell expression and synthesized by GenScript.
Transfect 1 million Sf9 cells in a 6-well plate with 2500 ng purified bacmid DNA mixed with FuGENE HD transfection reagent.
Ten days post-transfection, the first viral stock (V0) was harvested and used to infect 40 mL of Sf9 cells at 1×10⁶ cells/mL. After 4 days of expression at 27 °C , harvest cells by centrifugation at 2000 rpm for 00:15:00 .
15m
Upon the first signs of cytopathic effect (viability drop and yellow fluorescence), the culture was centrifuged, and the supernatant containing the amplified virus (V1) and used to generate V2 virus by adding 1 mL of V1 to 30 mL of Sf9 cells at 1×106 cells/mL. 
After three days 20 mL of V2 was used to infect 2 L of Sf9 cells (1×106 cells/mL). 
Cells were harvested when viability dropped to 90–95% (usually after five days after infection) washed with PBS, flash-frozen in liquid nitrogen, and stored at -80 °C until purification.
Cell Lysis
4h 15m
Thaw Frozen cell pellets On ice and resuspend in 50 mL ice-cold lysis buffer.
Perform resuspension by gentle pipetting, followed by mechanical lysis using a Dounce homogenizer:

  1. 10 strokes with loose pestle
  2. 2 × 20 strokes with tight pestle
Clarify Lysates by centrifugation at 9000 rpm for 00:15:00 (Sorvall RC6+ centrifuge, F21S rotor).
15m
Subject the supernatant to ultracentrifugation at 40000 rpm (~186,000 × g) for 01:00:00 at 4 °C (Beckman XPN90, Ti45 rotor).
1h
Resuspend the resulting membrane pellet in lysis buffer supplemented with 0.3% DDM using a Dounce homogenizer.
Solubilize membranes by incubation for 02:00:00 at 4 °C with gentle rolling.
2h
Remove insoluble debris by another round of ultracentrifugation at 40000 rpm for 01:00:00 at 4 °C .
1h
HisTrap Purification
Load the cleared DDM-solubilized supernatant onto a 1 mL HisTrap HP column (Cytiva, Cat# 17524802) pre-equilibrated in water and elution buffer.
Wash the column, and the protein elite using a stepwise gradient of imidazole (80, 150, 200, and 300 mM) in elution buffer.
Identify peak fractions containing ATG9A by SDS-PAGE and Coomassie staining.
Pool elution fractions and concentrate using a 100 kDa MWCO Amicon Ultra centrifugal filter.
Size-Exclusion Chromatography (SEC)
Perform further purification using Superose 6 Increase 10/300 GL column equilibrated with water and SEC buffer.
Pool fractions containing ATG9A , aliquote, snap-frozen in liquid nitrogen, and store at -80 °C .