May 13, 2026
Detection of FLAG ULK1 and Endogenous ATG13 Interaction
- Yongjia Duan1
- 1University of California Berkeley
Protocol Citation: Yongjia Duan 2026. Detection of FLAG ULK1 and Endogenous ATG13 Interaction. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9qqrml3e/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: 316994
Keywords: FLAG ULK1, ATG13, affinity pulldown, protein interaction, cell lysis, detection of flag ulk1, endogenous atg13 interaction, interaction between flag ulk1, affinity pulldown assay, endogenous atg13, flag ulk1, pulldown assay, atg13, using affinity
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Abstract
To detect the interaction between FLAG ULK1 and endogenous ATG13 using affinity pulldown assays.
Materials
NP-40 Lysis Buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) - 10 mL
Protease Inhibitor Cocktail (1X solution (e.g., Roche Complete)) - 1 mL
Phosphatase Inhibitor Cocktail (1X solution (e.g., Sigma)) - 1 mL
Anti-FLAG M2 Affinity Beads (30 μL per sample (MRC-PPU)) - 30 μL
2X LDS Sample Buffer (Life Technologies, #NP0007) - 40 μL
DTT (1 mM final concentration) - 1 μL
Beta-Mercaptoethanol (1 μL) - 1 μL
Spin-X® Column (Sigma, #CLS8162) - 1 unit
Reagents:
Buffer NP-40 Lysis Buffer - A non-ionic detergent used for cell lysis.
Inhibitor Protease Inhibitor Cocktail - Prevents proteolytic degradation of proteins.
Inhibitor Phosphatase Inhibitor Cocktail - Prevents dephosphorylation of proteins.
Reagent DTT - Reducing agent used in protein analysis.
Reagent Beta-Mercaptoethanol - Reducing agent used in protein analysis.
Troubleshooting
Problem
Low protein yield
Solution
Ensure complete lysis and proper incubation time with beads.
Problem
High background
Solution
Increase wash stringency or reduce incubation time with beads.
Safety warnings
Wear gloves and goggles when handling reagents and samples. Follow institutional safety protocols for handling biological materials and hazardous reagents.
Cell Lysis and Preparation
Collect cell lysates using NP-40 lysis buffer supplemented with protease and phosphatase inhibitors.
Cell Lysis
1. Wash cells with cold PBS (1 mL per well). 2. Add 200 μL of NP-40 lysis buffer supplemented with 1 mL of protease inhibitor cocktail and 1 mL of phosphatase inhibitor cocktail to each well. 3. Incubate on ice for 30 minutes, vortexing every 10 minutes. 4. Centrifuge at 14,000 rpm for 10 minutes at 4°C to collect supernatant.
FLAG Pulldown
Perform FLAG pulldown using anti-FLAG M2 affinity beads.
Incubation with Beads
1. Transfer 0.6 mg of cell lysate to a clean tube. 2. Add 30 μL of anti-FLAG M2 affinity beads. 3. Incubate for 4 hours at 4°C with end-over-end rotation (25 rpm).
Washing Beads
1. Centrifuge the tube at 1,000 g for 1 minute to pellet beads. 2. Remove supernatant and wash beads three times with 1 mL of NP-40 lysis buffer.
Elution of Proteins
1. Resuspend beads in 40 μL of 2X LDS buffer, 1 mM DTT, and 1 μL of beta-mercaptoethanol. 2. Heat at 95°C for 5 minutes to elute proteins.
Separation of Beads
1. Pass the eluate through a Spin-X® column to separate beads from the eluted proteins.
Protocol references