High-Density Antigen Microarrays for the Assessment of Antibody Selectivity

The success of affinity proteomic methods is dependent on well-characterized affinity binders. Antibodies are the classic affinity binders, and the availability of antibodies is constantly increasing. However, their suitability for a given proteomic method is often context-dependent and requires evaluation of their binding selectivity. One powerful proteomic platform for characterizing the binding selectivity of antibodies is the planar antigen microarray. In this chapter, we describe our in-house developed procedures for generating high-density planar antigen microarrays with an antigen content of 42,100 unique protein fragments. We provide details regarding the assay protocol used for the assessment of the binding selectivity of polyclonal antibodies produced by the Human Protein Atlas.