Reagents of Co-IP Protocol

• PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH to 7.4.
• Cell lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, sterile-filtered, add protease inhibitor cocktail before use.
• 3×SDS buffer：150mM Tris-HCl (pH6.8), 6%(W/V) SDS, 0.3%(W/V) BPB, 30% glycerol, 3% β-mercaptoethanol
• Antibody

Steps of Co-IP Protocol

Harvest and Wash Cells

1. Transfer the cultured cells from the culture dish to a 15-mL conical tube.

2. Centrifuge at 500×g for 2 min at 4°C and remove the supernatant.

3. Wash with ice-cold PBS and centrifuge at 500×g for 2 min at 4°C. Remove the supernatant.

4. Repeat Step 3 twice.

Cell Lysates Preparation

5. Resuspend the cell pellet in ice-cold cell lysis buffer (1 mL per 1 × 10⁷ cells) and incubate on ice for 10 min.

6. Sonicate cells in ice bath three times for 5 second pulses each.

7. Centrifuge at 13,000 × g at 4°C for 10 min, and transfer the supernatant to a fresh tube. Store the tube on ice for further use, or for long storage at -80°C.

Pre-wash the Magnetic Beads

8. Resuspend the magnetic beads by pipetting up and down for several times.

9. Transfer 20 μL of bead slurry to a fresh tube. Place the tube in a magnetic separation rack for seconds. Carefully remove the supernatant once the solution is clear.

10. Add 200 μL of cell lysis buffer (without protease inhibitor) to wash the magnetic bead pellet, pipette up and down for several seconds. Place the tube back in magnetic separation rack. Magnetize beads and remove the supernatant as dry as possible.

11. Repeat Step 10 twice

Pre-clear the Lysate (Optional)

Note: Pre-clearing the lysate is recommended to reduce the non-specific binding. However, if the protein is detected by western blotting, pre-clearing may not be necessary unless a contaminating protein is interfering with visualization of the protein of interest.

12. Add 200 μL cell lysate to 20 μL of pre-washed magnetic beads.

Note: The volume of cell lysate depends on the expression level of the protein of interest. A starting concentration between 250 μg/mL-1.0 mg/mL is recommended.

13. Incubate for 20 min at room temperature with gentle agitation.

14. Pellet beads out from the lysate by a magnetic separation rack, carefully collect the pre-cleared cell lysate, and discard the magnetic bead pellet.

Immunoprecipitation

15. Add relevant antibody to the pre-cleared cell lysate. Incubate for 30 min at room temperature or overnight at 4°C with gentle agitation to form the immunocomplex.

16. Pre-wash the magnetic beads as described in Pre-wash the Magnetic Beads (Step 8 to Step 11).

17. Transfer the lysate and antibody solution (immunocomplex) obtained in Step 15 to the tube containing the pre-washed magnetic bead pellet.

18. Incubate for 30 min at room temperature keeping gentle agitation.

19. Pellet beads using magnetic separation rack and discard the supernatant.

20. Wash pellet with 500 uL cell lysis buffer (without protease inhibitor). Magnetize beads and remove the supernatant as dry as possible.

21. Repeat Step 20 four times.

Elution

Note: There are three methods that can be used to elute the protein from the beads: SDS buffer elution, glycine buffer elution and urea buffer elution. Each of them have their own advantages. Here we describe an elution method based on SDS buffer which is highly efficient.

22. Resuspend the pellet with 50 μL SDS buffer, pipette up and down for several times to mix the sample.

23. Boil the sample for 5 min.

24. Pellet beads using magnetic separation rack. Transfer the supernatant to a fresh tube for further analysis.