Revision 2
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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Product Information

Product Usage Information

Use with Cell Signaling Technology’s PTMScan® kit protocol from the Immunoaffinity Purification (IAP) step.  Because the optimal amount of PTMScan® Control Peptides Phospho-Akt (RXXS*/T*) for each user’s experiments will depend on unique factors, such as mass spectrometer sensitivity, users may dilute these control peptides as needed.

1. Aliquot PTMScan® Control Peptides Phospho-Akt (RXXS*/T*) for storage as single-use units at -20°C or proceed to immediate usage.  
2. Resuspend sample peptides in the appropriate buffer and volume, e.g., 1.4 mL of PTMScan® IAP Buffer (1X).
3. Clear sample peptides by centrifugation.
4. Transfer clarified sample peptides to tubes containing IAP beads.
5. Add 10 µL of PTMScan® Control Peptides Phospho-Akt (RXXS*/T*) to IAP beads and sample peptides and mix well. 
6. Continue with PTMScan® or PTMScan® HS workflows at the 2-hour incubation step.
7. Detect PTMScan® Control Peptides Phospho-Akt (RXXS*/T*) in the LCMS data file.

Storage

This product is stable for 12 months when stored at -20ºC. Aliquot to avoid multiple freeze/thaw cycles.

Product Description

The PTMScan® Control Peptides Phospho-Akt (RXXS*/T*) enable quality control of immunoaffinity enrichment performance using PTMScan® or PTMScan® HS workflows.  These synthetic peptides contain a specific post-translational modification (PTM) that can be enriched by the associated PTMScan® or PTMScan® HS immunoaffinity purification (IAP) beads, as well as a stable heavy isotope that can be distinguished from endogenous peptides by the mass spectrometer. 

Background

Akt plays a central role in mediating critical cellular responses, including cell growth and survival, angiogenesis, and transcriptional regulation (1-3). 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 (4,5). Akt, p70 S6 kinase, and RSK additionally share specificity for Arg at position -5 (recognition sequence RXRXXS/T) (6). In a recent phosphoproteomic study (7) co-authored by scientists in the Cell Signaling Technology Site Discovery Group, over 300 downstream substrates for AGC-family kinases recognizing the RXRXXS/T motif were identified with PTMScan® Technology using Phospho-Akt substrate antibodies. These Cell Signaling Technology 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*/T*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the RXXS*/T* motif (S* = phospho-serine, T* = phospho-threonine).

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

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PTMScan is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。