Product Pathways - Screening Technologies
PTMScan® Phospho-Akt Substrate Motif mAb 2 (RXRXXS*/T*) Kit #5563
| No. | Size | Ordering Information | |
|---|---|---|---|
| 5563S | 1 Kit ( 5 assays ) |  |
ptmscan@cellsignal.com |
| Products Included | Quantity | Cap Color |
|---|---|---|
| PTMScan® (RXRXXS*/T*) 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.
Western Blotting
Akt substrate motif western blot analysis of extracts from MKN45 cells, untreated or Met inhibitor Su11274-treated, usingPhospho-Akt Substrate Motif (RXRXXS*/T*) Rabbit mAb.
Chart
Chart showing the proportions of underlying Akt substrate-like sequence motifs found in a PTMScan® study using Akt substrate motif mAb 2 and Orbitrap LC-MS analysis. Analysis of one experiment on peptides from H3255 cells gave 121 non-redundant sites containing Akt substrate-like motifs. The primary motifs are highlighted in white. The proportion containing phosphoserine was 72%, and the proportion with arginine residues at both the -5 and -3 positions was 65%. This antibody has a strong preference for arginine residues at the -5 and -3 positions, and to a lesser extent recognizes peptides with an arginine residue at just the -3 position.
Motif Logo
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.
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 DiscoveryGroup 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).
- 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.
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.
