A dot-blot-staining method for detecting phosphoproteins with a Phos-tag Aqua fluorescent dye

We describe a method for detecting phosphoproteins by dot-blot staining with a Phos-tag Aqua fluorescent dye. By using the method, three types of in vitro protein kinase assays for visualizing Tyr-, His-, and Asp-phosphoproteins, respectively, were performed. The staining procedure, which requires less than 2.5 h to complete, is conducted under conditions of neutral pH that are particularly advantageous for detecting labile Hisand Asp-phosphoproteins. This method promises rapid and easy detection of phosphoproteins, and should be useful in high-throughput profiling of in vitro kinase activities or in vitro kinase inhibition.