Top : Molecular Biology : Protein : Immunoprecipitation (IP)

Summary: Immunoprecipitation (IP) is a method that uses the antigen-antibody reaction principle to identify a protein that reacts specifically with an antibody from mixture of proteins so that its quantity or physical characteristics can be examined. A typical immunoprecipitation experiment contains the following steps. 1) The proteins from the cell or tissue homogenate are precipitated in an appropriate lysis buffer containing an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate (Protein A or G binds to the antibody, which is bound to its antigen) or a secondary antibody-agarose conjugate. 2) The immune complex is then captured on a solid support to which either Protein A or Protein G has been immobilized. Proteins that bind to the antibody are precipitated and proteins that do not are washed away. 3) Components of the bound immune complex (both antigen and antibody) are eluted from the support. 4) Immunoprecipited proteins are further analyzed by SDS-PAGE and immunoblotting to determine their molecular weights, to study their interactions with other proteins, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins.
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