Jun 15, 2026
Analytical Size-Exclusion Chromatography of ATG2A–WIPI4 Complex
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Justyna Sawa-Makarska 2026. Analytical Size-Exclusion Chromatography of ATG2A–WIPI4 Complex. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11oeovr2/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: August 13, 2025
Last Modified: June 15, 2026
Protocol Integer ID: 224621
Keywords: ASAPCRN, exclusion chromatography of atg2a, wipi4 complex formation, wipi4 complex, exclusion chromatography, wipi4 complex this protocol, wipi4, atg2a, western blot detection
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes how to analyze ATG2A–WIPI4 complex formation using analytical size-exclusion chromatography followed by Western blot detection.
Materials
SEC Buffer:
| A | B | |
| HEPES, pH 7.5 | 25 mM | |
| NaCl | 300 mM | |
| TCEP | 1 mM |
Protocol materials
anti-ATG2AProteintechCatalog #23226-1-AP
anti-HISQiagenCatalog #34660
Protein Sample Preparation
Prepare a protein mixture containing 4 micromolar (µM) ATG2A and 4.8 micromolar (µM) WIPI4 in SEC buffer to a final volume of 50 µL .
Incubate the protein mixture on ice for 02:00:00 to allow complex formation.
For control runs, prepare separate samples of ATG2A only and WIPI4 only at the same concentrations and volume.
SEC Run
Inject 50 µL of the protein mixture or control sample onto the equilibrated Superose 6 Increase 3.2/300 column (Cytiva, Cat# 29091598). Pre-equilibrated with performed with SEC buffer.
- Collect 100 µL fractions throughout the elution.
- Record the chromatogram for later comparison between mixture and controls.
Western Blot Analysis
Analyze collected fractions by Western blot. Detect ATG2A (anti-ATG2AProteintechCatalog #23226-1-AP ) and HIS-tagged WIPI4 ( anti-HISQiagenCatalog #34660 ) using specific antibodies.