A robotics-based automated assay for inorganic and organic phosphates

Phosphate analyses are fundamental to a broad range of biochemical applicat ions involving inorganic phosphate and organic phosphoesters such as phosph olipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric me thods of phosphate analyses based on formation of a phosphomolybdate comple x and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > cr ystal violet > quinaldine red > ascorbate reduction > antimony-modified asc orbate reduction, although only a fourfold improvement was observed in goin g from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest s ensitivity. However, where color stability and low blanks are more importan t than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a roboti c liquid-handling system substantially reduces the labor required to proces s large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-re sistant 96-well plate was developed to extend the assay to phosphate esters . The robotic-based assay was demonstrated with inorganic phosphate and a c ommon phospholipid, phosphatidylcholine. (C) 1999 Academic Press.