PTMScan^® Phospho-Akt Substrate Motif mAb 2 (RXRXXS*/T*) Kit #5563

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. A detailed protocol and Limited Use License allowing the use of the patented PTMScan^® method is included with the kit.

Storage

Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.

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, p70S6 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, p70 S6 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^® (RXRXXS*/T*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the (RXRXXS*/T*) motif (S* = phospho-serine, T* = phospho-threonine).

1. Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
2. Marte, B.M. and Downward, J. (1997) Trends Biochem Sci 22, 355-8.
3. Jiang, B.H. et al. (2000) Proc Natl Acad Sci USA 97, 1749-53.
4. Scheid, M.P. and Woodgett, J.R. (2000) Curr Biol 10, R191-4.
5. Montminy, M. (1997) Annu Rev Biochem 66, 807-22.
6. Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol 200, 62-81.
7. Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.
8. Moritz, A. et al. (2010) Sci Signal 3, ra64.