Product Pathways - Screening Technologies
PTMScan® Phospho-ST*P Motif (ST*P) XP® Kit #5566
The rabbit monoclonal antibody contained in this kit was developed using XMT®.
|5566S||1 Kit ( 5 assays )||email@example.com|
|Products Included||Quantity||Cap Color|
|PTMScan® (ST*P) 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.
Plk bing StP motif western blot analysis of extracts from HeLa cells, untreated or Nocodazole treated, using Phospho-ST*P Motif (ST*P) XP® Rabbit mAb.
Chart showing the proportion of underlying sequence motifs found in a PTMScan® study using a PLK binding motif antibody and OrbiTrap MS analysis. Analysis of peptides from HeLa cells, untreated and nocodazole-treated, gave 452 non-redundant sites containing a PLK binding and related motifs. The primary motif is highlighted in white 98% of these sites fit the ST*P motif; only 2% of the sites are from other phospho-Ser/Thr containing peptides that do not have the motif, which shows that this antibody is very specific for the ST*P motif.
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.
The ST*P motif is implicated in signaling by polo-like kinases (PLKs). These Ser/Thr protein kinases play essential roles during the cell cycle. At least four PLKs exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4 (2). PLKs have a highly conserved N-terminal kinase domain and a relatively divergent C-terminal domain called the Polo-box domain (PBD). Of the four PLKs, PLK1 is the best characterized (3). PLK1 functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody, and is crucial for proper progression through multiple stages of mitosis (4-6).The PBDs of PLK1 function as a phospho-Ser/Thr-binding module, optimally recognizing the sequence motif S[S*/T*][P/X] (PLK1 binding motif) (6). Binding of phosphopeptides containing S[S*/T*][P/X] motif by the PBD in PLK1 relieves its inhibitory function on kinase activity (7). S[S*/T*]P peptides are phosphorylated by proline-directed kinases such as cyclin dependent kinases (CDKs) (6). These findings imply that priming phosphorylations on substrates or docking proteins by other mitotic kinases such as CDKs may target PLK1 to its substrates and simultaneously activate its kinase domain. The ST*P Motif (ST*P) XP® Rabbit mAb contained in this kit, which binds to ST*P, was developed by Cell Signaling Technology and is a useful tool to study PLK1 binding proteins and PLK1 substrates.In this assay, PTMScan® (ST*P) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the ST*P motif (T* = phospho-threonine).
- Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
- Nigg, E.A. (1998) Curr Opin Cell Biol 10, 776-83.
- Donaldson, M.M. et al. (2001) J Cell Sci 114, 2357-8.
- Barr, F.A. et al. (2004) Nat Rev Mol Cell Biol 5, 429-40.
- Kishi, K. et al. (2009) Mol Cell Biol 29, 3134-50.
- Elia, A.E. et al. (2003) Science 299, 1228-31.
- Elia, A.E. et al. (2003) Cell 115, 83-95.
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For Research Use Only. Not For Use In Diagnostic Procedures.