Serine/Threonine Phosphatase Assay System
The Non-Radioactive Phosphatase Assay Systems provide a fast, convenient and flexible alternative for measuring protein phosphatase activity. These systems determine the amount of free phosphate generated in a reaction by measuring the absorbance of a molybdate:malachite green:phosphate complex. These systems allow the use of a variety of buffer conditions and substrates, including naturally phosphorylated proteins or synthetic phosphopeptides. The Serine/Threonine Phosphatase Assay System contains the chemically synthesized phosphopetide, RRA(pT)VA, a peptide substrate that is compatible with several serine/threonine phosphatases such as the Protein Phosphatases 2A, 2B, and 2C. However the supplied phosphopeptide is a poor substrate for Protein Phosphatase 1 because of its more stringent structural requirements. The Tyrosine Phosphatase Assay System contains two chemically synthesized phosphopeptides, END(pY)INASL and DADE(pY)LIPQQG, that serve as substrates for many protein tyrosine phosphatases. The effective range for the detection of phosphate released during an assay using the Phosphatase Assay Systems is 100-4,000pmol of phosphate. In addition to measuring phosphatase activity in partially fractionated and purified samples, the Phosphatase Assay Systems can also measure phosphatase activity in crude cell or tissue extracts. For this application, the high concentration of phosphate in these preparations is eliminated prior to performing the assay using the supplied Spin Columns, which rapidly and effectively remove free phosphate and other low-molecular-weight inhibitors from the sample. In addition, a unique Molybdate Dye Additive that is combined with the Molybdate Dye Solution aids in the solubilization of proteins exposed to the acid conditions of the Molybdate Dye Solution, which alone could potentially cause precipitation of the proteins.