- Co-Immunoprecipitation (Co-IP)
Co-Immunoprecipitation is a classical method researching protein interactions based on the specificity of antigen-antiody reaction. It helps determine whether two proteins interact or not in physiological conditions. Its principle is:
After cells are completely lysed under non-denaturing conditions, some proteins binding with each other are kept. Therefore if you use anti-X to precipitate protein X through Co-IP, then you can get other proteins that originally bind protein X in cells.
This method is applied to test whether two known proteins bind each other in cells, or to find a new protein that interacts with a known protein.
- Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation is an important tool to research interactions of DNA and proteins. Its principle is:
Cross-linking of target protein and DNA is performed by adding formaldehyde to growing cells, and chromatin is prepared, sheared by sonication, and precleared to reduce nonspecific immunoprecipitation. Immunoprecipitation is performed with a specific antibody. After elution of the protein-DNA complexes from protein A- or protein G-agarose resin, the samples are heated to reverse the covalent cross-links. The DNA fragments are purified and analyzed by PCR or real-time PCR.
- RNA immunoprecipitation
RNA immunoprecipitation is used to research interactions of RNA and proteins. Its principle is:
Precipitate the protein that physiologically binds RNA, then isolate the RNA out of antibody-protein A/G beads complex. Finally, use gene chip or other techniques to analyze the RNA.