Pierce™ Phosphoprotein Phosphate Estimation Assay Kit (Thermo Scientific™)

The Thermo Scientific Phosphoprotein Phosphate Estimation Kit enables classification and identification of proteins as phosphorylated (serine and threonine), as well as semi-quantitative assessment of the phosphorylation level.

Features of the Phosphoprotein Phosphate Estimation Kit:

• Specific—measures phosphoserine (p-Ser) and phosphothreonine (p-Thr) only; does not measure phosphotyrosine (p-Tyr)
• Convenient—test tube and 96-well microplate protocols included and require less than 90 minutes to perform
• Semi-quantitative—calculate moles of phosphate (P) per mole of protein using the included phosvitin standard
• Customizable—format is adaptable to development of specific assays for pure phosphoproteins when previously characterized standards are available

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 is completed in about one hour.

Applications:
• Identify proteins as containing phosphoserine or phosphothreonine phosphorylations
• Estimate the amount of pS and pT phosphorylation
• Develop specific quantitative assays for well-characterized phosphoproteins of interest

The assay can be used to identify whether a purified protein contains either phospho-serine (p-Ser) or phospho-threonine (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 phosvitin, a phosphoprotein 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 (1) not a phosphoprotein or (2) 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.