Product Pathways - Screening Technologies
PTMScan® Phospho-MAPK/CDK Substrate Motif (PXS*P and S*PXK/R) Kit #4652
|4652S||1 Kit (5 assays)||In Stockfirstname.lastname@example.org|
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|Products Included||Quantity||Cap Color|
|PTMScan® Phospho-MAPK/CDK Substrate Motif (PXS*P, S*PXK/R) Immunoaffinity Beads||10 x 80 µl||Blue|
|PTMScan® IAP Buffer (10X)||10 x 0.6 ml||White|
|PTMScan® Limited Use License|
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.
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 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.
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.
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.
PTMScan® is a trademark of Cell Signaling Technology, Inc.
UbiScan® is a trademark of Cell Signaling Technology, Inc.
AcetylScan® is a trademark of Cell Signaling Technology, Inc.
MethylScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, 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.