Chart showing the proportions of underlying sequence motifs found in a PTMScan® study using T*PP motif antibody and OrbiTrap MS analysis. The primary motif is highlighted in white. Analysis of peptides from HeLa cells, untreated and nocodazole-treated, gave 236 non-redundant sites. 88% of these sites fit motif T*PP; 8% sites are from motif T*P, and 4% of the sites are from motif S*P.
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.
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 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.
A T*PP phosphorylation motif has been extracted from a large-scale proteomic data set on HeLa nuclei (2). Western blot and proteomic analysis shows that phosphorylation on this motif is sensitive to cell cycle control. Similar motifs are phosphorylated by MAPK and CDK families of serine/threonine protein kinases, which play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (motifs PX(S/T)P and (S/T)PXK/R) (3-5). The T*PP Motif (T*PP) XP® Rabbit mAb contained in this kit, which binds to the motif T*PP, was developed by CST for use in the study and discovery of new kinase substrates.
In this assay, a PTMScan® (T*PP) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the T*PP motif (T* = phospho-threonine).
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.
PTMScan is a trademark of Cell Signaling Technology, Inc.
UbiScan is a trademark of Cell Signaling Technology, Inc.
XP is a registered 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 firstname.lastname@example.org.