Product Pathways - Screening Technologies
PTMScan® Phospho-MAPK/CDK Substrate Motif (PXS*P and S*PXK/R) Kit #4652
| No. | Size | Ordering Information | |
|---|---|---|---|
| 4652S | 1 Kit ( 5 assays ) |  |
ptmscan@cellsignal.com |
| Products Included | Quantity | Cap Color |
|---|---|---|
| PTMScan® (PXS*P or S*PXK/R) Motif Antibody Bead Conjugate | 5 x 80 µl | |
| PTMScan® IAP Buffer (10X) | 5 x 0.6 ml | White |
| PTMScan® Limited Use License |
PTMScan® Services
View sample data from a PTMScan® study using the Motif Antibody in this kit, provided in a PTMScan® Service report.
Directions for Use
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® 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. An alternate PTMScan® IAP Buffer Plus Detergent #9992 which may reduce nonspecific interactions is available separately. A detailed protocol and Limited Use License allowing the use of the patented PTMScan® method is included with the kit.
Description
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology 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® 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® services, please visit www.cellsignal.com/services/index.html.
Chart
Chart showing the proportions of underlying sequence motifs found in a PTMScan® study using a MAPK/CDK substrate motif antibody and OrbiTrap MS analysis. Analysis of peptides from HeLa cells, untreated (experiment #10684) and nocodazole-treated (experiment #10685), gave 439 nonredundant sites containing MAPK/CDK substrate and related motifs. 20% of these sites are motif PXS*PX(R/K); 70% are motif PXS*P; and 10% are motif S*PX(R/K). The antibody is phospho-serine specific and does not recognize phospho-threonine peptides.
Motif Logo
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.
Background
The MAPK and CDK families of serine/threonine protein kinases play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (2-4). MAPK phosphorylates substrates with the consensus sequence PX(S/T)P, and CDKs phosphorylate substrates containing the consensus sequence (S/T)PXK/R.Phospho-MAPK/CDK Substrates (PXSP or SPXR/K) Rabbit mAb #2325, contained in this kit, was developed at CST for use in the study and discovery of new MAPK and CDK substrates (5,6).In this assay, PTMScan® (PXS*P or S*PXK/R) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the PXS*P motif or the S*PXK/R motif (S* = phospho-serine).
- Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
- Cross, T.G. et al. (2000) Exp Cell Res 256, 34-41.
- Reynolds, C.H. et al. (2000) J Neurochem 74, 1587-95.
- Seger, R. and Krebs, E.G. (1995) FASEB J 9, 726-35.
- Holmes, J.K. and Solomon, M.J. (1996) J Biol Chem 271, 25240-6.
- Songyang, Z. et al. (1996) Mol Cell Biol 16, 6486-93.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
For Research Use Only. Not For Use In Diagnostic Procedures.
