Reverse Phase Protein Arrays

Reverse phase protein arrays (RPPA) are used to quantify proteins and protein posttranslational modifications in cellular lysates and body fluids. RPPA technology is suitable for biomarker discovery, protein pathway profiling, functional phenotype analysis, and drug discovery mechanism of action. The principles of RPPA technology are (a) immobilizing protein-containing specimens on a coated slide in discrete spots, (b) antibody recognition of proteins, (c) amplification chemistries to detect the protein-antibody complex, and (d) quantifying spot intensity. Construction of a RPPA begins with the robotic liquid transfer of protein-containing specimens from microtiter plates onto nitrocellulose-coated slides. The robotic arrayer deposits each sample as discrete spots in an array format. Specimens, controls, and calibrators are printed on each array, thus providing a complete calibrated assay on a single slide. Each RPPA slide is subsequently probed with catalyzed signal amplification chemistries and a single primary antibody, a secondary antibody, and either fluorescent or colorimetric dyes. The focus of this chapter is to describe RPPA detection and imaging using a colorimetric (diaminobenzidine (DAB)) detection strategy.