Microarray analysis of Akt PH domain binding employing synthetic biotinylated analogs of all seven phosphoinositide headgroup isomers

► We have developed assays employing microarray analysis using 96-well microplates and robotic pin printing onto glass slides for high-throughput analysis of protein-phosphoinositide binding interactions. ► For these assays, we designed and synthesized biotin-conjugates corresponding to the headgroups of all seven naturally occurring phosphoinositide isomers. ► Binding analysis was performed by immobilizing synthetic phosphoinositide-conjugates onto surfaces followed by antibody-based detection of protein binding. ► These platforms were applied to study the phosphoinositide-binding specificity of the PH domain of Akt and understand the effects of ligand surface density on protein binding.