This chart shows the underlying CK2 substrate-like motifs in a PhosphoScan® LC-MS/MS experiment of tryptic peptides generated from Jurkat cells treated with Calyculin A #9902 and pervanadate, and immunoprecipitated using PTMScan® Phospho-CK2 Substrate Motif (S*/T*DXE) Immunoaffinity Beads. Of the 354 motif-containing peptides, 54% contained phospho-serine and 46% contained phospho-threonine. The proportion of peptides with aspartate or glutamate at the +1 position is 83% and at the +3 position is 73%. Peptides with aspartate or glutamate at both the +1 and +3 positions is 63%.
The Motif Logo was generated from a PhosphoScan® LC-MS/MS experiment using 471 nonredundant tryptic peptides derived from Jurkat cells treated with Calyculin A #9902 and pervanadate, and immunoprecipitated using PTMScan® Phospho-CK2 Substrate Motif (S*/T*DXE) Immunoaffinity Beads. This PhosphoSitePlus® logo represents the enrichment of an amino acid at a residue position relative to the abundance of that amino acid in the background, in this case, the human proteome. Residues represented at the top of the logo are the most enriched, whereas those below the y-axis occur less frequently in the sample than would be expected in the proteome. The PSP logo reflects the specificity of the antibody for the S*/T*DXE motif. For more information on motif analysis using PSP, please visit www.phosphosite.org.
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 are 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.
PhosphoScan® Technology employs a phospho-residue (Tyr, Ser, Thr) motif antibody for phospho-peptide immunoaffinity purification from cell extracts combined with LC tandem MS/MS to identify and quantify changes in phosphorylation levels (1). Casein Kinase II (CK2) is a highly conserved, ubiquitously expressed, and constitutively active tetrameric serine/threonine protein kinase with hundreds of substrates participating in the regulation of a variety of cellular processes including cell cycle progression, apoptosis, transcription, inflammation, and the DNA damage response. Research studies have implicated CK2 in roles related to viral infection, cancer and other diseases (2-6). CK2 substrates contain multiple acidic residues (Asp and Glu) located downstream of the phosphorylated serine or threonine residue. The consensus sequence for CK2 substrates is pS/pTD/EXD/E with the most crucial residue at the +3 position, followed by the residue at the +1 position (7).