Two-Step Co-Immunoprecipitation (TIP) Enables an Efficient and Highly Selective Isolation of Native Protein-Complexes

Co-immunoprecipitation (co-IP) is the most frequently used technique for the isolation of protein-protein (PPI) or protein-nucleic acid interactions (PNI) under native expression conditions. A major disadvantage of co-IP is the abundance of non-specific binders that can impede downstream applications. To overcome this limitation, we developed the two-step co-IP (TIP) that significantly improves the purification of native protein-containing complexes. TIP can be applied with a broad range of specific mono- and polyclonal antibodies. Using TIP we purified IKKalpha- and CD95- interacting proteins in primary human T cells, detected all major binders by mass spectrometry analysis and identified two novel CD95-associated proteins.