SH2 Domain-Based Tyrosine Phosphorylation Array

Tyrosine phosphorylation plays a major role in intracellular signal transduction pathways. Phosphorylated tyrosine residues initiate signaling pathways by recruiting proteins containing Src homology-2 (SH2) domains. Herein is described a high-throughput assay to detect interactions between phosphorylated proteins and human SH2 domains, in order to profile the genome-wide phosphorylation status of cells under a variety of biological conditions. The SH2 domain array was prepared by immobilizing 115 SH2 domain proteins on a membrane. The array assay is straightforward with no expensive equipment or radioactivity required: the cell lysate is incubated with the array membrane, and the signal is measured using a chemiluminescence-based detection system.