Highly sensitive detection of protein phosphorylation by using improved P hos‐tag B iotin

We have previously shown that the dinuclear zinc(II) complex P hos‐tag and its derivatives act as phosphate‐capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more‐advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized P hos‐tag derivatives, including a bisbiotinylated P hos‐tag ( BTL ‐108), a tetrakisbiotinylated P hos‐tag ( BTL ‐109), and a monobiotinylated P hos‐tag with a dodeca(ethylene glycol) spacer ( BTL ‐111), as well as the commercially available product BTL ‐104. Among these complexes, BTL ‐111 showed the best performance in W estern blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz‐crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel P hos‐tag sensor chip coated with BTL ‐111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL ‐104. Moreover, a peptide microarray technique using the ECL system and BTL ‐111 permitted high‐throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.