1. Background and purpose of the procedure

This immunoprecipitation protocol details individual steps for the enrichment and purification process of proteins from cell lysate using an antibody bound to agarose or magnetic beads. Purified immunoprecipitated proteins can be eluted and the resultant targets can be analyzed and/or identified using several assays, including ELISA, immunoblot, or MS/MS.

2. Materials and equipment

• Specific antibody for the protein target (preferably IgG)
• Lysate sample
• Lysis buffer (see 3. Recipes)
• Pure Proteome Protein A/G Mix Magnetic Beads (Merk, LSKMAGAG10)
• 1X Laemmli buffer containing DTT (see 3. Recipes)
• Chaotropic buffer (see 3. Recipes)

• Ice/Ice bucket
• Vacuum trap or aspiration
• Magnetic stand for use with magnetic Protein A/G beads
• 1.5 mL or 2 mL microcentrifuge tubes
• Microcentrifuge (refrigerated, 4°C)
• Rotating platform (refrigerated, 4°C)

3. Recipes

2X Lysis Buffer

30 mM HEPES (Fisher BioReagents, BP310-1 – stock solution @ 1 M) [600 µL for 10 mL]
100 mM KCl (Fisher Chemical, AR Certified, P-4280-53 – stock solution @ 1 M) [2 mL for 10 mL]
20 mM NaCl (Fisher Chemical, AR Certified, S-3160-53 – stock solution @ 5 M) [80 µL for 10 mL]
2 mM MgCl₂ [6▪H₂O] (Fisher Chemical, M-0550-53 – stock solution @ 1 M) [40 µL for 10 mL]
5% Glycerol (Fisher BioReagents, Molecular Biology Grade, BP2294) [1 mL for 10 mL]
ddH₂O qsp 10 mL [6.3 mL for 10 mL]

2X lysis buffer can be prepared in 100 mL batches and then aliquoted (5 or 10 mL in 15 mL centrifuge tubes) before being stored at -20°C for up to a year.

1X Lysis Buffer

2X lysis buffer [10mL for 20 mL]
0.5% n-Dodecyl-β-Maltoside (DDM) (Anatrace, D310-5 – stock solution @ 5%) [2 mL for 20 mL]
5 µM GDP (Sigma, G-7127 – stock solution @ 1 mM) [100 µL for 20 mL]
5 mM NaF (Fisher Chemical, AR Certified, S-3920-60 – stock solution @ 100 mM) [1 mL for 20 mL]
1 mM NaVO₄ (Thermo Scientific Chemicals, 081104.22 – stock solution @ 1 M) [20 µL for 20 mL]
1X Complete Protease Inhibitor (Stock solution @ 25X [1 tablet + 2 mL ddH₂O]) [800 µL for 20 mL]
ddH₂O qsp 20 mL [6.1 mL for 20 mL]

1X lysis buffer can be stored at 4°C for up to 2 weeks.

Alternatively, NaF can be replaced with 1 µM Microcystine (Alexis, ALX-350-012-C500 – stock solution @ 1 mM) [20 µL for 20 mL]

1X Laemli Buffer containing DTT

Laemmli 4X (Bio-Rad, 1610747) [200 µL for 800 µL]
10% DTT (Sigma-Aldrich, 646563-10X.5mL) [2 µL for 800 µL]
ddH₂O qsp 800 µL [698 µL for 800 µL]

Chaotropic Buffer (Urea Elution Buffer)

8 M Urea (Fisher Chemical, Biochemical Grade, U-P610-50) [4.8 g for 10 mL]
20 mM Tris-HCl (Fisher Chemical, T-P631-60 – stock solution @ 1 M) [200 µL for 10 mL]
100 mM NaCl (Fisher Chemical, AR Certified, S-3160-53 – stock solution @ 5 M) [200 µL for 10 mL]
ddH₂O qsp 10 mL

Adjust pH to 7.5. Chaotropic buffer can be stored at 4°C for up to 2 weeks.

4. Methods

1. Wash the beads for lysate pre-clearing by adding lysis buffer to the magnetic beads and vortex for 20 seconds [200μL for 5 μL of beads]
2. Place the microcentrifuge tube in the magnetic stand and wait until all the beads stick to the side of the tube and remove all the liquid. Be careful not to disturb the beads
3. Add whole cell lysate to the protein A/G beads and vortex for 20 seconds [5 μL of beads for 1 mL of whole cell lysate]
4. Incubate for 30 minutes at 4 ˚C on a rotating platform
5. Insert the tube into the magnetic stand and transfer the pre-cleared lysate into a clean 1.5 mL microcentrifuge tube
6. Add the antibody to the pre-cleared lysate sample. Typically, 0.5-1.0 μg of purified target-specific antibody is added to 0.5-1.0 mL of cell lysate
7. [Optional] If the primary antibody displays a low affinity for Protein A or G, addition of a secondary anti-immunoglobulin antibody is recommended, add 0.5–1.0 μg of anti-immunoglobulin antibody after 30 min.
8. Incubate overnight (>18h) at 4 ˚C on a rotating platform
9. Wash the beads for antibody-antigen complexes capture by adding lysis buffer to the magnetic beads and vortex for 20 seconds [600 μL for 10 μL of beads]
10. Place the microcentrifuge tube in the magnetic stand and wait until all the beads stick to the side of the tube and remove all the liquid. Be careful not to disturb the beads
11. Add antibody-antigen lysate to the protein A/G beads and vortex for 20 seconds [10 μL of beads for 1 mL of antibody-antigen lysate]
12. Incubate for 4 hours at 4 ˚C on a rotating platform
13. Insert the tube into the magnetic stand and remove the supernatant. Be careful not to disturb the beads
14. Wash the immune complexes five times using lysis buffer to remove nonspecific binding. Remove the final wash as completely as possible. Allow the remaining buffer from the sides of the tube to collect at the bottom of the tube for 1 min before removing it a second time
15. The captured antigen can be eluted by incubation in 1X Laemmli buffer containing DTT for subsequent detection by immunoblot [150 μL for 10 μL of beads]
16. [Alternative] For subsequent detection by MS/MS use a chaotropic buffer (urea-based) to detach the antigen from the antibody.
    • Ensure all the residual supernatant is removed from the beads.Add chaotropic buffer to the beads and vortex [50-80 μL for 10 μL of beads]
    • Incubate at room temperature with frequent agitation for 30 min.
    • Transfer the supernatant to a 1.5 mL microcentrifuge tube
    • Repeat the steps above twice to ensure the entire captured complexes have been released from the beads

5. References

DeCaprio J, Kohl TO. Immunoprecipitation. Cold Spring Harb Protoc. 2017. doi: 10.1101/pdb.prot098640. PMID: 29196601.

Abcam. Immunoprecipitation (IP) protocol.

6. Revision history

Revision #	Date	Prepared by
1.0	2023-09-07	Elie Besserer-Offroy
Summary of modifications
Initial version of the protocol

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Please cite this protocol and associated references in your publications.
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