This chart shows the underlying motif distribution in a PhosphoScan® LC-MS/MS experiment using 750 nonredundant tryptic peptides generated from mitotic HeLa cells. HeLa cells were treated with thymidine (2 mM, 18 hr) followed by Nocodazole #2190 (60 nM, 24 hr) and immunoprecipitated using PTMScan® Phospho-Ser-Pro-Pro Motif [pSPP] Immunoaffinity Beads.
The Motif Logo was generated from a PhosphoScan® LC-MS/MS experiment using 750 nonredundant tryptic peptides derived from mitotic HeLa cells. HeLa cells were treated with thymidine (2 mM, 18 hr) followed by Nocodazole #2190 (60 nM, 24 hr) and immunoprecipitated using PTMScan® Phospho-Ser-Pro-Pro Motif [pSPP] Immunoaffinity Beads. The logo demonstrates the relative frequency of amino acids in a given position around the modified site 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 -20ºC. 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 www.cellsignal.com/common/content/content.jsp?id=ptmscan-services.
The MAPK and CDK families of serine/threonine protein kinases play important roles in cell signaling and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (1-6). To facilitate the study and discovery of new MAPK and CDK substrates, Cell Signaling Technology has developed antibodies that bind to phospho-threonine or phospho-serine followed by proline.
The S/TPP motif matches the consensus sequence for targeted phosphorylation by Cdc2 (7,8), ERK1, ERK2 (9), SAPK/JNK, CDK5, and GSK3 (10).
AcetylScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MethylScan is a trademark of Cell Signaling Technology, Inc.
PhosphoScan is a trademark of Cell Signaling Technology, Inc.
PTMScan is a trademark of Cell Signaling Technology, Inc.
UbiScan is a trademark of Cell Signaling Technology, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.