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.
As an integral part of the machinery of cellular function, proteins undergo regulation by a variety of post-translational modifications. One of the most prevalent and widely studied PTMs is Serine/Threonine phosphorylation. A few prominent kinases targeting a handful of substrate consensus motifs account for a majority of the tens of thousands of known and predicted sites on more than 13,000 human proteins (1-3). Cell Signaling Technology has developed phospho-Ser/Thr motif antibodies for proteomic profiling of kinase substrates. These include, among others, substrates of Akt, AMPK, MAPK, CDK, PKA, PKC, and CKII.
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