Thermo Scientific Pierce Phosphoprotein Staining Kit is for specific, colorimetric, in-gel detection of phosphoserine and phosphothreonine residues in phosphorylated proteins.
The Pierce Phosphoprotein Staining Kit is a colorimetric stain to specifically detect proteins separated by polyacrylamide gel electrophoresis (SDS-PAGE) that are phosphorylated at serine and/or threonine residues. Staining is achieved by first hydrolyzing the phosphoprotein-phosphosphoester linkage using 0.5N NaOH in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resulting insoluble nitrophospho-molybdate complex is finally stained with a solution of the basic dye, methyl green. The reagents in this kit hydrolyze the phosphoester linkage of phosphoserine and phosphothreonine. Phosphotyrosine is not hydrolyzed and cannot be detected with this kit. Although the staining method is not sufficiently sensitive to detect subtle changes in phosphorylation of low-abundance proteins, the kit provides a convenient system for assessing purification and phosphorylation status of phosphoproteins during preparative workflows.
- Specific for pSer and pThr phosphoproteins – for use with SDS-PAGE to detect abundant serine- and threonine-phosphorylated proteins in the sample
- Easy-to-follow protocol – produces results in approx. three hours, yielding phosphoproteins that are identified as green to green-blue bands
- Qualitative staining – detection limit depends on the extent of phosphorylation and protein size and abundance; detects 40 to 80ng phosvitin and 80ng to 160ng beta-casein per band
- Controls included – complete kit includes one positive control protein (phosvitin) and one negative control protein (soybean trypsin inhibitor) for method validation
- Compatible with double-staining –phosphoprotein-stained gels can be secondarily stained with Thermo Scientific GelCode Blue Stain Reagent (Part No. 24590) for total protein profiling
- Seven solutions (3L total), plus 2 vials of lyophilized protein (1mg each)
- Evaluate the progress of a phosphoprotein purification process
- Identify approximate molecular weights of most abundant phosphoproteins in a sample
|Protocol summary for the Thermo Scientific Pierce Phosphoprotein Staining Kit.
|Specific detection of phosphorylated proteins with the Thermo Scientific Pierce Phosphoprotein Staining Kit. An identical selection of proteins was run on two gels and stained with either the Phosphoprotein Stain (left) or Thermo Scientific GelCode Blue Stain for total protein detection (right). Arrows denote lanes containing phosphoproteins. A: soybean trypsin inhibitor (negative control), B: bovine serum albumin, C: phosvitin, D: histone III-S, E: ovalbumin, F: β-casein and G: prestained protein molecular weight markers. Note that coomassie dye does not detect heavily phosphorylated proteins, such as phosvitin (right, lane C).
The limit of detection of this stain depends on several factors, including the molar amount of phosphate loaded onto the gel and the accessibility of these groups within the electrophoresed sample to hydrolysis during the staining procedure. For several proteins, the calculated lower molar limit of phosphate detection varied more than five-fold and, therefore, lower limits of detection must be determined empirically for each phosphoprotein.
- Simpson, R.J. (2003). Proteins and Proteomics. A Laboratory Manual. Cold Spring Harbor Laboratory Press. pp. 201-202.
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