|5636||PTMScan® Phospho-Tyrosine Mouse mAb (P-Tyr-100) Kit||
PhosphoScan® Kit (P-Tyr-100) from Cell Signaling Technology (CST) allows for the purification and identification of tyrosine phosphorylation sites in cellular proteins when coupled with LC tandem mass spectrometry using CST's patented PhosphoScan® technology. The assay is based on the specific enrichment of phosphotyrosine-containing peptides using an antibody against phosphotyrosine. Cells are lysed in a urea-containing buffer, and cellular proteins are digested by protease and fractionated by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using Phosphotyrosine Mouse mAb (P-Tyr-100) #9411 coupled to protein G agarose beads*. Overnight incubation ensures high-affinity binding of phosphotyrosine-containing peptides to P-Tyr-100 beads. Unbound peptides are removed through washing, and phosphotyrosine-containing peptides are eluted with dilute acid. Reversed-phase chromatography is performed on microtips to separate phosphopeptides from antibody and to concentrate them for LC tandem mass spectrometry. The kit also contains control peptides from 2x108 Jurkat cells that were treated with pervandate and calyculin A. These control peptides allow the investigator to determine the efficiency of the immunoaffinity purification (IAP) step and the LC-MS/MS analysis (1 assay). *Product is specific to this kit.
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology for peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. These include phosphorylation (PhosphoScan®), ubiquitination (UbiScan®), acetylation (AcetylScan®), and methylation (MethylScan®), among others. PTMScan® enables researchers to isolate, identify and quantitate large numbers of post-translationally modified cellular peptides with a high degree of specificity and sensitivity providing a global overview of PTMs in cell and tissue samples without preconceived biases about where these modified sites occur. For more information on PTMScan® services, please visit www.cellsignal.com/services/index.html
Tyrosine phosphorylation plays a key role in cellular signaling (2). In cancer, unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals (3). Current phosphoproteomic approaches generally reveal only small numbers of tyrosine phosphorylation sites, in keeping with the low level of phosphotyrosine relative to phosphoserine and phosphothreonine residues (4). PhosphoScan® Kit, based on CST's published method (1,5), isolates large numbers of cellular phosphotyrosine-containing peptides. This allows the investigator to obtain a global overview of tyrosine phosphorylation in the cell, covering many classes of proteins without preconceived biases about where tyrosine phosphorylation sites will be found.
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