PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads (#20432) Datasheet Without Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Information

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.

Product Description

PTMScan^® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads employ immobilized metal affinity chromatography for capturing phosphorylated peptides. Negatively charged phosphate groups are attracted to the positively charged metal ions on the beads. In conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS), this approach enables researchers to isolate, identify, and quantitate large numbers of phosphorylated cellular peptides with a high degree of specificity and sensitivity, providing a global overview of phosphorylation in cell and tissue samples. For more information on PTMScan^® Proteomics Services, please visit https://www.cellsignal.com/services/index.html.

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 Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺ are often used to purify proteins rich in poly-Histidine or Cysteine as well as proteins with metal affinity. Trivalent metal ions, Fe³⁺, Ga³⁺, Al³⁺, as well as Ti⁴⁺ and Zr⁴⁺ 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. Ga³⁺ and Fe³⁺ are comparable with respect to the number of phosphopeptides identified (2). Compared to metal oxide affinity chromatography (MOAC) using TiO₂, Fe³⁺ IMAC performed marginally better with TiO₂ having a preference for acidic phosphopeptides (pI > 4) relative to Fe³⁺ 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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PhosphoScan is a registered trademark of Cell Signaling Technology, Inc.

PTMScan is a registered trademark of Cell Signaling Technology, Inc.

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Limited Uses

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