Introduction to the principle and steps of co-immunoprecipitation (Co-IP)

one. principle

Co-Immunoprecipitation is a classical method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction of two proteins in intact cells. The principle is that when the cells are lysed under non-denaturing conditions, many protein-protein interactions within the intact cells are preserved. If X is immunoprecipitated with an antibody to protein X, the protein Y bound to X in the body can also be precipitated. At present, prorein A is pre-bound and solidified on the argarose beads to react with the antigen-containing solution and antibody, and the prorein A on the beads can adsorb the antigen. This method is often used to determine whether two target proteins bind in vivo; it can also be used to identify new interaction partners for a particular protein.

Advantages :

(1) The interacting proteins are all modified after translation and are in a natural state;

(2) Protein interactions are carried out in a natural state to avoid artificial effects;

(3) The interacting protein complex in a natural state can be isolated.

Disadvantages:

(1) Low affinity and transient protein-protein interactions may not be detected;

(2) The combination of the two proteins may not be a direct combination, and there may be a third party acting as a bridge in the middle;

(3) It is necessary to predict what the protein of interest is before the experiment to select the antibody to be finally detected. Therefore, if the prediction is not correct, the experiment will not get results, and the method itself is risky.