PTMScan® Phospho-Akt Substrate Motif mAb 1 (RXXS*/T*) Kit #5561
Additional Information
This product is intended for peptide enrichment and mass spectrometry analysis. To learn more about our Proteomics Kits and Services please answer a few questions for our Proteomics group.
Product Information
Product Usage Information
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.
Storage
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Protocol
Product Description
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.
Background
Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (2-4). It is a member of an important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, which includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, p70 S6 kinase, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (5,6). Akt, p70S6 kinase and RSK additionally share specificity for Arg at position -5 (recognition sequence RXRXXS/T) (7) In a recent phosphoproteomic study (8) co-authored by scientists in the CST Site Discovery Group over 300 downstream substrates for AGC family kinases recognizing the RXRXXS/T motif were identified with PhosphoScan Technology using Phospho-Akt substrate antibodies. These CST™ antibodies are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.In this assay, PTMScan® (RXXS
- Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
- Marte, B.M. and Downward, J. (1997) Trends Biochem Sci 22, 355-8.
- Jiang, B.H. et al. (2000) Proc Natl Acad Sci USA 97, 1749-53.
- Scheid, M.P. and Woodgett, J.R. (2000) Curr Biol 10, R191-4.
- Montminy, M. (1997) Annu Rev Biochem 66, 807-22.
- Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol 200, 62-81.
- Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.
- Moritz, A. et al. (2010) Sci Signal 3, ra64.
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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
AcetylScan is a registered trademark of Cell Signaling Technology, Inc.
MethylScan is a registered trademark of Cell Signaling Technology, Inc.
PTMScan is a registered trademark of Cell Signaling Technology, Inc.
UbiScan is a registered trademark of Cell Signaling Technology, Inc.
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