Jun 16, 2026

Pull-Down Assay to Assess ATG2A Binding to WIPI4 and LC3B

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz9bw5gx1/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.rm7vz9bw5gx1/v1
Protocol Citation: Elisabeth Holzer 2026. Pull-Down Assay to Assess ATG2A Binding to WIPI4 and LC3B. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9bw5gx1/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 13, 2025
Last Modified: June 16, 2026
Protocol  Integer ID: 224626
Keywords: ASAPCRN, down assay to assess atg2a, assess atg2a, lc3b this protocol, atg2a, western blot detection, wipi4, down assay, lc3b, assay
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes how to assess ATG2A binding to WIPI4 and LC3B using a FLAG-based pull-down assay followed by SDS–PAGE and Western blot detection.
Materials
SEC300:
AB
HEPES, pH 7.525 mM
NaCl300 mM
TCEP1 mM
SEC500:
AB
HEPES, pH 7.525 mM
NaCl500 mM
TCEP1 mM
Protocol materials
anti-ATG2AProteintechCatalog #23226-1-AP
anti-HISQiagenCatalog #34660
Binding assay
Incubate beads coated with 3×FLAG-tagged ATG2A (5 µL slurry per condition) with 5 micromolar (µM) purified 6×HIS-tagged WIPI4, LC3B, or GFP (negative control) in SEC300 buffer.

Incubate for 01:00:00 at room temperature with gentle rotation.

Washing and fraction collection
Wash once with 100 µL SEC300 buffer. Add 40 µL SEC300 buffer to the beads.

Split the sample into two equal parts:
  • Low-salt fraction – store on ice for analysis (see step 6)
  • High-salt fraction 
For the high-salt fraction perform the following washes (100 µL each):
  • 2× washes with SEC300 buffer
  • 1× wash with SEC500 buffer
  • 2× washes with SEC300 buffer

Resuspend beads of low- and high-salt fraction in 20 µL SEC300 and mix with SDS loading buffer.

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.