This chart shows the underlying motif distribution in a PhosphoScan® LC-MS/MS experiment using 180 nonredundant tryptic peptides derived from Jurkat cells treated with Calyculin A #9902 (10 nM, 20 min) and Pervanadate (1 mM, 20 min) and immunoprecipitated with PTMScan® Phospho-PDK1 Docking Motif [(F/Y)p(S/T)(F/Y)] Immunoaffinity Beads.
The motif logo was generated from a PhosphoScan® LC-MS/MS experiment of 180 nonredundant tryptic peptides derived from Jurkat cells treated with Calyculin A #9902 (10 nM, 20 min) and Pervanadate (1 mM, 20 min) and immunoprecipitated with PTMScan® Phospho-PDK1 Docking Motif [(F/Y)p(S/T)(F/Y)] Immunoaffinity Beads. The logo demonstrates the relative abundance of amino acids in a given position around the centrally phosphorylated residue within this data set.
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. For more information on PTMScan® Proteomics Services, please visit https://www.cellsignal.com/services/index.html.
A hallmark of signal transduction pathways is the reversible phosphorylation of serine and threonine residues within recurring sequences, or motifs, in target proteins. These are not limited to consensus sequences that are substrates for protein kinases (1), but also those that are recognized by phosphorylation-dependent binding proteins such as 14-3-3 (2). Phosphoprotein interacting domains are critical elements in regulating intracellular communication. For example, Akt, a kinase that regulates cell survival, is activated by phosphorylation at Ser473, a site preceded by Phe at -4 and -1 and followed by Tyr at +1 (3). Phosphorylation at this and similar sites on RSK2, p70 S6 kinase and certain PKC isoforms is required for their binding to 3-phosphoinositide-dependent kinase 1 (PDK1) and subsequent downstream signaling (4-7). Through these interactions, PDK1 is involved in the regulation of key cellular processes including proliferation, differentiation and apoptosis. Cell Signaling Technology has developed phospho-Ser/Thr motif antibodies as powerful tools for proteomic profiling of kinase substrates and phosphoprotein binding domains.