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Immunoprecipitation: Definition, Principle and Procedure

Immunoprecipitation (IP)
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.

Immunoprecipitation IP Procedure
Summarised Procedure
1.  Suitable antibody against the target antigen or protein is added
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 90–100ยบ 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.


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