Coimmunoprecipitation

Overview

Immunoprecipitation of intact protein complexes is referred as co-immunoprecipitation. Co-immunoprecipitation works effectively when the proteins involved have the ability to tightly bind with one another. This helps in pulling out multiple members of the complex by latching onto one member by using an antibody. Usually, this is orchestrated when one protein in the complex is known and a suitable antibody is used. By targeting the known antibody, it becomes easier to pull the entire protein complex from the solution. For the same reason, the technique is sometimes referred as pull-down immunoprecipitation. The technique is primarily used by molecular biologists to study protein-protein interactions.

Principle

Co-Immunoprecipitation shares the same principle of antigen-antibody specificity/reaction as in any Immunoprecipitation technique. The known protein in the complex is referred as bait protein and the interacting protein is known as prey protein respectively.

Applications

  • Prove interaction between two proteins
  •  Identify a new protein by using a known one.

Advantages

  • Post-translationally modified and conformationally natural.
  • Proteins interact in almost physiological condition.

 Disadvantages

  • Weaker signals from low affinity proteins are not detected
  • Antibody selection is critical and target protein prediction needs to be correct
  • third protein in certain protein-protein interaction could weaken the procedure

 Protocol

  1. Cultured cells are carefully washed with chilled PBS twice.
  2. Lysis buffer is added (1ml for 100 cells)
  3. The cells are scrapped off from 1.5ml Eppendorf tubes with a clean and cold scraper. It is incubated in low-speed rotating shaker for 15 mins at 4°C.
  4. Centrifuge at 14,000 g 4°C for 15min, the supernatant is transferred to new tubes immediately.
  5. the protein A/G-agarose beads are washed twice with PBS and a 50% protein A/G agarose working solution (in PBS) is made.
  6. 50% protein A/G agarose is added with ratio of 100μl for a 1ml sample solution.
  7. Incubate in horizontal shaker for 10min at 4°C. This done mainly to eliminate non-specific binding proteins.
  8. Again, Centrifuge 14,000g at 4°C for 15min.
  9. the supernatant is transferred to new tubes and discard protein A/G-agraose beads.
  10. The protein is quantified by BCA assay or other methods.
  11. The protein is diluted to 1μg/μl with PBS to decrease the concentration of detergents.
  12. primary antibody is added at an appropriate amount to 500μl total volume.
  13. the antigen-antibody complex is incubated for overnight at 4°C in a rotating shaker.
  14. Centrifuge 14,000g for 5s, keep the pellet and wash with pre-chilled washing buffer for 3 times. (800μl each)
  15. The supernatant is collected and analysis is carried out via SDS-PAGE, western-blot, or mass spectra analysis.

Note: This protocol gives lower yield, but avoids the problem of co-elution of antibodies. If higher yield is expected, the antibody can be mixed with protein sample prior to addition of Protein A/G-agarose beads. The antibodies can co-elude with target protein and interference can be observed in western blot detection.

Preparation of Reagents and Buffers

  1. PBS
  2. RIPA is made of Tris-HCl: 50 mM, pH 7.4, Nonidet P-40 (NP-40): 1%, Deoxycholate Na:25%,
  3. NaCl: 120 mM, EDTA: 1 mM, PMSF: 1 mM, Leupeptin 1 μg/ml, Aprotinin 1 μg/ml, Pepstatin1 μg/ml, Na3VO4: 1 mM, and NaF: 1 mM.
  4. protein A/G-agarose beads
  5. Specific antibody (MAb or PAb)

Frequently Asked Questions

  1. What are the different types of immunoprecipitation?

  • Individual protein immunoprecipitation
  • protein complex immunoprecipitation
  • chromatin immunoprecipitation
  • RNP Immunoprecipitation
  1. What are the different methods of immunoprecipitation?

Direct method: The beads bound to antibodies are added to the protein mixture and the target proteins are captured by the beads through antibodies, or simply immunoprecipitated. The antibodies are immobilized on solid phase substrate such as microscopic agarose and superparamagnetic microbeads.

Indirect method: Antibodies are added to protein complex and bind to target proteins. Beads coated with protein A/G is added subsequently to target bound proteins in the complex. Beyond this point, the protocols converge and as the end-results are one and the same.

  1. What is chromatin immunoprecipitation?

Chromatin immunoprecipitation is used to study the relationship between proteins and DNA. The technique combines DNA sequencing techniques with immunoprecipitation to identify the binding sites of DNA-associated proteins.

  1. What is RNP Immunoprecipitation?

RNP immunoprecipitation targets ribonucleoproteins; the cells are lysed. The target protein and associated RNA are immunoprecipitated using antibody to target protein of interest. RNA-protein complexes are separated and in the identity of RNA is determined using cDNA sequencing.

  1. What are tagged proteins?

One inherent disadvantage in immunoprecipitation is the difficulty in generating antibody for a known protein. To counter this disadvantage, research groups often design tags focused on C or N terminal end of the target protein. Examples of tags include green fluorescent protein, Glutathione S transferase and the Flag tag.