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|