ANYL 127 |
| Here, we describe an efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on IMAC using a phosphate-binding tag molecule (a dinuclear zinc(II) complex). The binding, washing, and elution processes were all conducted without detergents or reducing agents at pH 7.5. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an EGF-stimulated A431 cell lysate. The total time for the chromatography (1 mL gel scale) was less than one hour. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis. |
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General Papers
7:00 PM-9:00 PM, Sunday, 10 September 2006 Moscone Center -- Hall D, Poster
Division of Analytical Chemistry |