IP is a technique used to enrich or purify a specific protein from a complex mixture using an antibody.
During IP, an antibody (monoclonal or polyclonal) is allowed to form an immune complex with the antigen it recognizes, typically a protein present in a suspension (e.g. cell lysate). The immune complex is then captured on a solid support (e.g.agarose or sepharose) carrying immobilized Protein A or Protein G. After washing away any proteins not bound by the immobilized Protein A or G, the components of the immune complex(antibody and antigen) are eluted and analyzed by SDS-PAGE, often followed by Western blot analysis (WB) to visualize the protein of interest.
Polyclonal antibodies | Monoclonal antibodies |
Inexpensive to produce | Expensive to produce |
Skills required are low | Training is required for the technology used |
Time scale is short | Time scale is long for hybridomas |
Produces large amounts of non-specific antibodies | Can produce large amounts of specific antibodies |
Recognizes multiple epitopes on any one antigen | Recognizes only one epitope on an antigen |
Can have batch-to-batch variability | Once a hybridoma is made, it is a constant and renewable source |
- | No or low batch-to-batch variability |