Motif analysis was done using all tyrosine phosphopeptides generated from studies in the published report by Rikova, K.A., et al. (2007) Cell 131, 1190-1203. The Motif Logo, drawn from 4160 non-redundant sites, shows the amino acid distributions around the sites recognized by Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411. The motif analysis shows that this antibody is a general phospho-tyrosine antibody and does not have amino acid preferences at other positions.
Cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and the resulting peptides are purified by reversed-phase, solid-phase extraction. Peptides are then subjected to immunoaffinity purification using a PTMScan® Motif antibody conjugated to protein G agarose beads. Unbound peptides are removed through washing, and the captured PTM-containing peptides are eluted with dilute acid. Reversed-phase purification is performed on microtips to desalt and separate peptides from antibody prior to concentrating the enriched peptides for LC-MS/MS analysis. CST recommends the use of PTMScan® IAP Buffer #9993 included in the kit. A detailed protocol and Limited Use License allowing the use of the patented PTMScan® method is included with the kit.
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology (CST) 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® Technology 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 (1). For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/common/content/content.jsp?id=ptmscan-services.
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). Antibodies specific for phospho-tyrosine (4,5) have been invaluable reagents in these studies. The Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411, developed by Cell Signaling Technology, provides an exceptionally sensitive tools for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery.
In this assay, PTMScan® (Y*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing *Y motif (Y* = phospho-tyrosine).