Immunoprecipitation is a method that enables the purification of a protein or antigen.
Principle: is antigen –antibody interaction
Definition: It is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
2. As antigen-antibody interactions are highly specific, antibody binds to the target antigen and forms an antigen-antibody complex
3. This antigen-antibody complex is made insoluble by adding protein A or G beads
4. On centrifugation of solution, antigen-antibody complex pellet down and supernatant can be removed
5. Continue to pellet the beads with Ag-Ab complex in centrifuge between each wash. In order to minimize background, care should be given to remove the supernatant completely each time.
6. Resuspend the beads with Ag-Ab complex in loading buffer and mix gently
7. Boil at 90–100º C for 5–10 minutes to dissociate the Ag-Ab complex from the beads
8. Centrifuge the sample to pellet the beads
9. Collect the supernatant carefully and load onto an SDS-PAGE gel to separate proteins prior to Western blot analysis for detection of protein.
Detailed protocol @ http://www.leinco.com/immunoprecipitation