Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS

The ability to understand and characterize phosphorylation is important to the study of cell signaling and to synthetic biology approaches. Current methods for characterizing kinase-substrate interactions are limited by their inherently low throughput and the heterogeneity of samples analyzed. Recent advances in yeast surface display techniques provide new opportunities for studying individual kinase-substrate interactions in a stimulus-independent fashion. Here, we describe techniques for building substrate libraries into full-length domains of interest that, when co-localized intracellularly with individual kinases, result in the display of phosphorylated domains on the yeast surface, as well as fluorescence-activated cell sorting and magnetic bead selection techniques for enriching from these libraries based on phosphorylation state.