CST, 20432L, PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads

Affinity and Purification Reagents for studying in the research area. 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. 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. Storage 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. Background Immobilized metal affinity chromatography, or IMAC, has been widely used to enrich proteins and peptides from biological samples by binding to clusters of negative charge. Divalent transition metal ions Co , Cu , Ni , and Zn are often used to purify proteins rich in poly-Histidine or Cysteine as well as proteins with metal affinity. Trivalent metal ions, Fe , Ga , Al , as well as Ti and Zr are commonly used for phosphopeptide enrichment for proteomic studies (1). Iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) are used for chelating the metal ions to agarose-coated beads. In comparison studies, NTA has been shown to perform better than IDA at selectively capturing and identifying more phosphopeptides. Ga and Fe are comparable with respect to the number of phosphopeptides identified (2). Compared to metal oxide affinity chromatography (MOAC) using TiO , Fe IMAC performed marginally better with TiO having a preference for acidic phosphopeptides (pI > 4) relative to Fe which preferred less acidic peptides (pI < 4) (3). The PTMScan Fe-NTA Magnetic Beads offer an efficient tool for phosphopeptide enrichment with little or no bias for phospho-residue context. They can be employed independently or in conjunction with immunoaffinity based enrichment to complement any PhosphoScan LC-MS/MS proteomic study. Alternate Names Fe based enrichment; IMAC; immobilized metal; metal affinity; metal based enrichment; MOAC; phosphopeptide; phosphoproteome; pSTY; TiO2