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

Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIP(n)s), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIP(n) binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein-PIP(n) binding interactions is hindered by the complexity of the membrane environment and of the PIP(n) structures. Herein, we describe two rapid assays of use for characterizing protein-PIP(n) binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIP(n) headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P(3) and PI(3,4)P(2) as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIP(n) ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein-membrane binding interactions. Copyright Â