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. Phospho-peptides are then subjected to enrichment using PTMScan® IMAC Beads. Unbound peptides are removed through washing, and the captured phospho-peptides are eluted with basic pH buffer. Reversed-phase purification is performed on microtips to desalt and separate peptides from impurities prior to concentrating the enriched peptides for LC-MS/MS analysis. A detailed protocol is included with the beads.
IMAC Fe-NTA Magnetic Beads are supplied in 20% ethanol. For long term storage, replace the buffer with long term storage buffer (1:1:1 v/v/v of methanol, acetonitrile, and 0.01% acetic acid). See protocol for details. Store at 4ºC. Do not freeze the beads.
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