Abstract
In this chapter, we provide a standard protocol for phosphate-affinity column chromatography for the separation of phosphorylated and nonphosphorylated biomolecules by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties (Phos-tag Tip) was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphorylated biomolecules, such as phosphopeptides, in the field of neuroscience.
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Acknowledgments
This work was supported in part by KAKENHI Grants 25293005 to E.K., 15 K07887 to E.K.-K., and 26460036 to T.K.
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Kinoshita, E., Kinoshita-Kikuta, E., Koike, T. (2019). Enrichment of Low-Molecular-Weight Phosphorylated Biomolecules Using Phos-Tag Tip. In: Li, K. (eds) Neuroproteomics. Neuromethods, vol 146. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9662-9_7
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DOI: https://doi.org/10.1007/978-1-4939-9662-9_7
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