Representative motif analysis of 2362 phospho-tyrosine tryptic peptides generated from a PhosphoScan® experiment of Jurkat cells treated with Calyculin A #9902 and pervanadate using PTMScan® Phospho-Tyrosine Motif (Y*) (P-Tyr-1000) Immunoaffinity Beads. Motif logo was generated using the PhosphoSitePlus® Logo Generator and reflects relative frequency of residues in each position. For more information, please visit PhosphoSitePlus® at www.phosphosite.org.
Motif analysis was done using all tyrosine phosphopeptides generated from a PhosphoScan® experiment of mouse brain and mouse liver tryptic peptides using PTMScan® Phospho-Tyrosine Motif (Y*) (P-Tyr-1000) Immunoaffinity Beads. The Motif Logo, drawn from 1178 nonredundant sites, shows the amino acid distributions around the sites recognized by Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb #8954. The motif logo 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 A 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 are 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.
PhosphoScan® Technology employs phospho-residue (Tyr, Ser, Thr) motif antibodies for phosphopeptide immunoaffinity purification from cell extracts combined with LC tandem MS/MS to identify and quantify changes in phosphorylation levels (1). Tyrosine phosphorylation plays a key role in cellular signaling. Research studies have shown that in cancer, unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals (2). Antibodies specific for phospho-tyrosine have been invaluable reagents in these studies (3,4). The Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb #8954, developed by Cell Signaling Technology, provides an exceptionally sensitive tool for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery. PTMScan® (Y*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing Y* motif (Y*= phospho-tyrosine).