FIGURE 3.

In situ Binding Assays – Affinity Purification-Mass Spectrometry (AP-MS). (A) Native AP-MS. Cellular lysates containing the protein of interest (POI) are exposed to an antibody toward the POI. The resulting immunocomplexes are precipitated from the lysate by exposure to a Protein G-labeled matrix while the non-binding (NS) proteins are washed away. (B) GFP-Tagged AP-MS. The POI is expressed in cells as a fusion with green fluorescent protein (GFP). The cells are lysed; and the GFP-containing complexes precipitated by an anti-GFP antibody-labeled matrix while the NS proteins are washed away. (C) Tandem AP-MS (TAP-MS). The POI is expressed in cells as a fusion with two tags separated by a tobacco etch virus (TEV) protease site. The cells are lysed; and the lysate mixed with antibodies toward the distal tag (Tag1). The resulting immunocomplexes are precipitated from the lysate by exposure to a Protein G-labeled matrix while the non-binding (NS) proteins are washed away. The tag is then cleaved by adding TEV protease and the complexes precipitated by the proximal tag (Tag2) binding to a binding domain (BD)-labeled matrix.