May 13, 2026
Detection of ATG13-WIPI3 Interaction in Stable Cell Lines
- Yongjia Duan1
- 1University of California Berkeley
Protocol Citation: Yongjia Duan 2026. Detection of ATG13-WIPI3 Interaction in Stable Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqxxnxlk5/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
We use this protocol and it's working
Created: May 13, 2026
Last Modified: May 13, 2026
Protocol Integer ID: 316990
Keywords: ATG13, WIPI3, co-immunoprecipitation, SDS-PAGE, stable cell lines, wipi3 interaction in stable cell line, wipi3 in various stable cell line, detection of atg13, interaction of atg13, wipi3 interaction, various stable cell line, wipi3, stable cell line, atg13
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Abstract
To detect the interaction of ATG13 with WIPI3 in various stable cell lines using co-immunoprecipitation (Co-IP) and subsequent analysis.
Materials
10 cm culture dishes (Sterile, tissue culture treated) - 4
Co-IP lysis buffer (Pre-chilled, 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM EDTA, 5% glycerol, 1% NP-40) - 50 mL
EBSS (Earle's Balanced Salt Solution) - 50 mL
SDS loading buffer (2× concentration) - 10 mL
Lipofectamine 2000 (Transfection reagent (Thermo Fisher, cat. #11668019)) - 20 μL
pCAG-TSF-GFP-WIPI3 (Plasmid for transfection) - 10 μg
Pierce™ Anti-HA Magnetic Beads (For immunoprecipitation (Thermo, 88836)) - 100 μL
Reagents:
Buffer Co-IP lysis buffer - Buffer for cell lysis and protein extraction
Buffer SDS loading buffer - Buffer for protein denaturation
Protease Inhibitor Protease inhibitor cocktail - Inhibits protease activity during lysis
Troubleshooting
Problem
Low transfection efficiency
Solution
Optimize DNA:lipid ratio or use fresh reagents.
Problem
Poor protein yield
Solution
Ensure proper lysis conditions and check for protease activity.
Safety warnings
Use gloves and goggles when handling reagents and biological materials. Dispose of all waste according to institutional biosafety guidelines.
Cell Preparation
Seed ATG13-KO+Halo-LC3, ATG13-KO+Halo-LC3+HA-ATG13-WT, and ATG13-KO+Halo-LC3+HA-ATG13-(HF|DD) stable cell lines into 10 cm dishes at a density of 1 × 10⁵ cells/mL.
Transfection
Transfect cells with 10 μg of pCAG-TSF-GFP-WIPI3 plasmid using Lipofectamine 2000 at a DNA:lipid ratio of 1:2.
Treatment
Replace the medium with fresh medium 2 hours before treatment with EBSS for 1 hour.
Cell Lysis and Co-IP
Harvest cells using pre-chilled Co-IP lysis buffer.
Lysis
Add 1 mL of Co-IP lysis buffer to the cell pellet and incubate on ice for 1 hour.
Centrifugation
Centrifuge at 13,000 × g for 10 minutes at 4°C. Save 1/20 of the supernatant as input.
Immunoprecipitation
Incubate the remaining supernatant with 100 μL of Pierce™ Anti-HA Magnetic Beads at 4°C overnight.
Washing
Wash the bead-protein complexes five times with Co-IP lysis buffer, 10 minutes per wash.
Elution
Elute bound proteins by boiling in 2× SDS loading buffer for 10 minutes.
SDS-PAGE and Analysis
Resolve the eluted proteins by SDS-PAGE.
Gel Electrophoresis
Load 10 μL of eluted fractions onto a 15% polyacrylamide gel and run the gel according to standard protocols.