Thermo Scientific™ Pierce™ Phosphoprotein Phosphate Estimation Assay Kit

Description

• 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
• 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
• 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
• Highlights: 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 Includes: Ammonium molybdate solution, malachite green solution, Tris-buffered saline pack and phopvitin positive control Recommended for: 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