High-Throughput Post-Translational Modification Analyses by Reverse Phase Protein Array

Post-translational modifications play a crucial role in regulating protein functions by chemically modifying amino acids without altering underlying protein sequences. Protein modifications are highly involved in cellular signal transduction pathways that require swift changes between active and inactive states. Various methods, including reverse phase protein array (RPPA), have been developed to comprehensively assess the proteomic profile. RPPA technology, a robust antibody-based platform, can detect not only protein expression but also modifications, such as phosphorylation, methylation, and acetylation. This chapter presents the detailed RPPA protocol for profiling post-translational modifications, including protein phosphorylation using whole cell lysates and histone modifications using purified histones.