The chart shows the underlying distribution of phospho-peptide motifs in a PhosphoScan® LC-MS/MS experiment using 581 nonredundant Lys-C/tryptic peptides generated from 293T cells treated with Calyculin A #9902 (10 nM, 30 min) and immunoprecipitated with PTMScan® Phospho-Ser/Thr Motif [pS/T] Immunoaffinity Beads.
The Motif Logo was generated from a PhosphoScan® LC-MS/MS experiment using 581 nonredundant Lys-C/tryptic peptides generated from 293T cells treated with Calyculin A #9902 (10 nM, 30 min) and immunoprecipitated using PTMScan® Phospho-Ser/Thr Motif [pS/T] Immunoaffinity Beads. The Motif Logo demonstrates the relative abundance of amino acids in a given position 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.
As an integral part of the machinery of cellular function, proteins undergo regulation by a variety of post-translational modifications. One of the most prevalent and widely studied PTMs is Serine/Threonine phosphorylation. A few prominent kinases targeting a handful of substrate consensus motifs account for a majority of the tens of thousands of known and predicted sites on more than 13,000 human proteins (1-3). Cell Signaling Technology has developed phospho-Ser/Thr motif antibodies for proteomic profiling of kinase substrates. These include, among others, substrates of Akt, AMPK, MAPK, CDK, PKA, PKC, and CKII.
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