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.Learn more about how we get our images
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.Learn more about how we get our images
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.
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
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.
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).
AcetylScan is a trademark of Cell Signaling Technology, Inc. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. MethylScan is a trademark of Cell Signaling Technology, Inc. PTMScan is a trademark of Cell Signaling Technology, Inc. UbiScan is a trademark of Cell Signaling Technology, 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 firstname.lastname@example.org.
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