Immunoprecipitation (IP) is a protein biochemistry technique of pulling down a protein of interests out of solution containing thousands of soluble proteins, usually using solid substrate attached antibody against the protein. Co-Immunoprecipitation (Co-IP) refers to the protein complex immunoprecipitation. In Co-IP cells are lysed in mild nondenaturing lysis buffer in order to keep the protein complexes intact.
* Permeabilization solution
Triton X-100 (0.5%, w/v)
MgCl2 (3 mM)
NaCl (60 mM)
Sucrose (300 mM)
HEPES, pH6.9, (10 mM)
* Lysis buffer
Depending on the protein complex to be analyzed, different lysis buffer may be used for protein-protein interactions study. For other applications following nondenaturing cell lysis solution can be used to solubilize cellular proteins.
Triton X-100 (1%, w/v)
SDS (0.2%, w/v)
Sodium deoxycholate (0.5%, w/v)
NaCl (150 mM)
MgCl2 (1 mM)
EGTA (1 mM)
2-Mercaptoethanol (2-ME, 10 mM)
Tris-HCl, pH 7.4 (15 mM)
Protease inhibitor cocktails (PMSF, aprotinin, leupeptin, pepstatin A)
Note protease inhibitor cocktail is commercially available from several vendors.
* Laemmli sample buffer
Tris-HCl, pH 6.8 (0.0625 M)
General Immunoprecipitation Procedures:
1. Lyse cells in appropriate lysis buffer for immunoprecipitation at 4c.
2. Lysate preclearing: During preclearing step the Protein A/G agarose beads (without antibody coating) are added to the lysate. Proteins that non-specifically bind to the agarose beads will be precipitated and removed. Precleared sample lysate are now ready for incubation with primary antibody.
3. Primary antibody incubation: Add primary antibody against the protein of interests and incubate on ice (4C) for 1 hour or overnight with gentle agitation. Note the antibody should be 4-5 folds in excess over the antigene to make sure all antigen binds to antibody.
4. Protein A/G agarose binding of primary antibody: Wash the protein A/G agarose beads and add beads to the sample. Incubate for 30 min at 4C with gentle agitation. Pellete by centrifugation briefly at 4C.
5. Washing immunoprecipitate: The wash step will remove the nonspecific binding. Wash 3 times using washing buffer and precipitate by centrifugation.
6. Dissociation of protein complex from protein A/G beads: Add Laemmli sample buffer (or buffer containing SDS, 2-ME, or 8M Urea) to sample and heat denature at 90-100c. Vortex denatured sample. Remove the agarose beads pellet by centrifugation and analyze the supernatant on SDS-PAGE gel electrophoresis. Gel can be stained with Coomassie blue or analyzed by western blot.
Troubleshooting for immunoprecipitation:
No specific bands visible on coomassie blue-stained gel.
- Antigen abundancy is low. Use a more sensitive staining method such as silver stain and increase the starting protein quantity within the upper limit allowed.
- Antibody may not recognize the protein antigen.
- Molecular Biology