PTMScan^® Phospho-ST*P Motif (ST*P) XP^® Rabbit mAb Kit #5566

Product Usage Information

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.

Storage

Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.

Product Description

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.

Background

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).

1. Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
2. Nigg, E.A. (1998) Curr Opin Cell Biol 10, 776-83.
3. Donaldson, M.M. et al. (2001) J Cell Sci 114, 2357-8.
4. Barr, F.A. et al. (2004) Nat Rev Mol Cell Biol 5, 429-40.
5. Kishi, K. et al. (2009) Mol Cell Biol 29, 3134-50.
6. Elia, A.E. et al. (2003) Science 299, 1228-31.
7. Elia, A.E. et al. (2003) Cell 115, 83-95.