Phosphoprotein Phosphate Estimation Assay Kit

Phosphoprotein Phosphate Estimation Assay Kit: The Phosphoprotein Phosphate Estimation Assay Kit is designed to aid in characterization of the status and extent of phosphorylation of purified protein samples. The assay is based on the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and quantification of the released phosphate with malachite green and ammonium molybdate. The assay is easily performed in 96-well microplates or test tubes and can be completed in about one hour. The assay can be used to identify whether a purified protein contains either phospho-serine (p-Ser) or phosphothreonine (p-Thr), as well as to estimate the level of this type of phosphorylation. For quantitation, the test protein sample is compared with specific concentrations of our phosphopeptide standard, a phosphopeptide of known phosphorylation level. The alkaline hydrolysis step does not release phosphate from phospho-tyrosine (p-Tyr) residues in peptide linkage. Therefore, a negative result for an unknown purified protein preparation indicates that the protein is either not a phosphoprotein or is phosphorylated exclusively at tyrosine residues. In the latter case, Western blot analysis using an anti-phosphotyrosine antibody will be necessary to distinguish between these two possibilities. The kit is sufficient for 500 test tube assays or 2000 microplate assays.