The Magnetic Phosphopeptide Enrichment Kit uses Phospho Magnetic Beads to enhance recovery of phosphorylated peptides that might otherwise be difficult to purify and analyze. The kit can be used to rapidly enrich phosphopeptides containing phosphotyrosine, phosphoserine, or phosphothreonine. It is compatible with various elution buffers, to provide samples in formats that are ideal for different downstream applications such as matrix-assisted laser desorption ionization (MALDI) mass spectroscopy or liquid chromatography (LC).
Phospho Magnetic Beads bind phosphopeptides through the specific interaction of phosphate groups with immobilized ferric ions on the surface of the beads. Magnetic particles in the beads facilitate quick and easy separation of the bead-peptide complexes from solutions using either magnetic force or gravity. Any magnetic separator that accommodates 1.5 ml microcentrifuge tubes can be used. Alternatively, a microcentrifuge can be used to separate the particles.
Optimized Buffers Minimize Nonspecific Binding
The buffers in the kit are detergent-free and have been optimized for specific phosphopeptide binding and elution. The improved Binding/Wash Buffer minimizes the nonspecific binding of peptides containing acidic residues that can complicate immobilized metal affinity chromatography (IMAC) technologies. The kit includes 100 mM ammonium bicarbonate elution buffer (pH 9.0–9.5), however, bound phosphopeptides can also be eluted into other buffers, such as 100 mM sodium carbonate buffer (pH 9.3) or phosphate buffered saline, to accommodate different applications.
Nanoscale Purification in Small Elution Volumes
Phospho Magnetic Beads are supplied as a 5% suspension, and have a binding capacity of approximately 1–2 pmol of phosphate per µg of Phospho Magnetic Beads. Different amounts of beads may be used, depending on the initial sample concentration. Phosphopeptides can be eluted in volumes as small as 20 µl to yield concentrated samples.