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.
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).
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