Product Pathways - Screening Technologies
PTMScan® Phospho-PKA Substrate Motif (RRXS*/T*) Kit #5565
|5565S||1 Kit ( 5 assays )||email@example.com|
|Products Included||Quantity||Cap Color|
|PTMScan® (RRXS*/T*) Motif Antibody Bead Conjugate||5 x 80 µl|
|PTMScan® IAP Buffer (10X)||5 x 0.6 ml||White|
|PTMScan® Limited Use License|
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.
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.
Chart showing the proportions of underlying sequence motifs found in a PTMScan study using PKA substrate antibody and Orbitrap LC-MS analysis. Combined analysis of two experiments (#10619, #10620) on peptides from PC-12 cells gave 187 non-redundant sites containing PKA substrate and related motifs. The primary motifs are highlighted in white. The proportion containing phosphoserine was 82%, and the proportion with arginine residues at both the -3 and -2 positions (PKA substrate motif) was 64%. Although this antibody has a strong preference for the PKA substrate motif, it also recognizes peptides with just an arginine residue at the -3 position.
An important class of kinases, refered to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (2,3). Phospho-PKA substrate-specific antibodies from CST are powerful tools for investigating the regulation of phosphorylation by PKA and other Arg-directed kinases, as well as for high throughput kinase drug discovery.In this assay, PTMScan® (RRXS*/T*) Motif Antibody bead conjugates are used to specifically enrich phospho- peptides containing the RRXS*/T* motif (S* = phospho- serine, T*= phospho-threonine).
- Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
- Montminy, M. (1997) Annu Rev Biochem 66, 807-22.
- Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol 200, 62-81.
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For Research Use Only. Not For Use In Diagnostic Procedures.