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.
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.
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).
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.
XP is a registered 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 [email protected] For information regarding commercial licensing terms please contact CST Pharma Services Department at [email protected]