High-Throughput Evaluation of Metabolic Activities Using Reverse Phase Protein Array Technology

Reverse Phase Protein Array (RPPA) technology is a high-throughput proteomics platform capable of reliably assaying thousands of samples at the same time for hundreds of proteins, including for total protein expression and its post-translational modifications such as phosphorylation. In this chapter, the authors present detailed protocols on how to set up and apply RPPA technology for protein profiling on 230+ antibodies for total and phosphoproteins in various signaling pathways. RPPA technology includes antibody selection and validation, RPPA printing, antibody labeling, slide scanning, image analyses, data processing and quality control, and data analyses. The authors present details on using RPPA technology to understand the role of two-pore channels (TPCs) in cell signaling and metabolism in isolated primary mouse embryonic fibroblasts from wild type and TPC1/TPC2 double knockout mice. The RPPA analysis establishes important insights on proteins and signaling pathways regulated by TPCs.