CST, 71652S, PTMScan ® Phospho-PKC Substrate Motif [(R/K)(R/K)Xp(S/T)X(R/X)] Kit

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.
Storage
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Protocol
Available protocols: PTMScan
Background
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).