The chart shows the underlying motif distribution in a PhosphoScan® LC-MS/MS experiment using 431 nonredundant Lys-C digested peptides generated from mouse embryo and immunoprecipitated using PTMScan® Phospho-PKC Substrate Motif [(R/K)(R/K)Xp(S/T)X(R/X)] Immunoaffinity Beads. Peptides containing phospho-threonine comprised 30% of the data set while phospho-serine contributed 70%.
The Motif Logo was generated from 431 unique peptides identified in a PhosphoScan® LC-MS/MS experiment using Lys-C digested peptides derived from mouse embryo and immunoprecipitated with PTMScan® Phospho-PKC Substrate Motif [(R/K)(R/K)Xp(S/T)X(R/X)] Immunoaffinity Beads. The logo depicts the relative frequency of amino acids in each position surrounding the central phosphorylated residue.
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. For more information on PTMScan® Proteomics Services, please visit https://www.cellsignal.com/services/index.html.
Protein kinase C (PKC) family members are involved in a number of signal transduction processes including secretion, gene expression, proliferation, and muscle contraction and many PKC substrates continue to be unidentified (1,2). Isozymes of PKC are subdivided into conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). PKCα, βI, βII, and γ isoforms belong to the cPKC group (1). When activated, cPKC isozymes phosphorylate substrates containing Ser or Thr, with Arg or Lys at the -3, -2, and +2 positions, and a hydrophobic amino acid at position +1 (1-3).