Pull-down assays with purified proteins in vitro

In Vitro mTOR - Claspin Binding Assay Protocol

Abstract

Protein-protein interactions can be assessed by many procedures. Among them, immunoprecipitation with a specific antibody is one of the most commonly used methods. Here, we describe a method to investigate the interactions using purified proteins.

This protocol was designed to observe the interaction between Claspin and mTOR proteins using immunoprecipitation but should be applicable to the interactions of any proteins.

Keywords: protein-protein interactions, immunoprecipitation, antibody  

1. Reagents Preparation

10X Reaction Buffer (200 μL)

Mix the following components and keep on ice:

• 400 mM Hepes (pH 7.6): 80 μL (Stock: 1 M) 
• 200 mM K-Glutamate: 40 μL (Stock: 1 M) 
• 25 mM MgCl₂: 2.5 μL (Stock: 2 M) 
• 10 mM EGTA: 20 μL (Stock: 100 mM) 
• 0.1% Triton X-100: 20 μL (Stock: 10%) 
• 1 mM ATP: 2 μL (Stock: 100 mM) 
• 5 mM PMSF: 10 μL (Stock: 100 mM) 
• H₂O: 25.5 μL 
  • Place on a magnet and remove supernatant.
  • Wash the complex 3 times with 100 μL Washing Buffer. Resuspend gently during each wash.
  • Place on a magnet and remove supernatant from the final wash/transfer step.
  • Resuspend beads in 12.5 μL of 1x SB (Sample Buffer).
  • Boil the samples and proceed to SDS-PAGE analysis.

2. Reaction Setup

Total reaction volume is 50 μL. Prepare the samples as follows:

Instructions:

1. Mix components according to the table and spin down.
2. Incubate at 4°C for 60 minutes.

3. Input Preparation: Save 8 μL of the reaction mixture, add 2 μL of 5x SB (Sample Buffer), and boil for later analysis.

    

3. Beads-Antibody Binding 

1. Beads Wash: Take 25 μL Dynabeads®* and wash with Washing Buffer (CSK Buffer with 0.01% Tween-20). Use a magnet to remove the supernatant.
2. Antibody Binding: Add 5 μg of Antibody (anti-N-Claspin) diluted in 30 μL CSK Buffer containing 0.01% Tween-20 per sample.
3. Incubation: Incubate with rotation for 30 minutes at room temperature.
4. Wash: Remove supernatant using a magnet, wash with Washing Buffer, and resuspend for IP.

* Dynabeads; use the original solution supplied in 50% slurry. 25µl of solution should contain ~12.5 µl beads and ~12.5µl suspension solution.

4. Immunoprecipitation & Elution

1. Antigen Binding: Add the 42 μL reaction sample (from Step 2) to the beads-Ab complex.
2. Incubation: Incubate with rotation for 10 minutes at room temperature.
3. Washing:
4. Optional Transfer for Purity: Resuspend the complex in 50 μL Washing Buffer and transfer the bead suspension to a clean tube. (This is recommended to avoid coelution of proteins bound to the tube wall.)
5. Elution:

• 1x sample buffer: 62.5 mM Tris-HCl（pH6.8）, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol（もしくは        100 mM DTT）, 0.02% BPB
• Antibody: Affinity-purified IgG fractions from rabbit sera (1-2mg/ml)