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Enrichment of phosphorylated proteins from cell lysate using a novel phosphate‐affinity chromatography at physiological pH
Author(s) -
KinoshitaKikuta Emiko,
Kinoshita Eiji,
Yamada Atsushi,
Endo Mika,
Koike Tohru
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600252
Subject(s) - lysis , chromatography , chemistry , elution , tris , affinity chromatography , phosphate , agarose , lysis buffer , tcep , protein purification , biochemistry , enzyme , phosphine , catalysis
While phosphoproteins have attracted great interest toward the post‐genome research ( e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate‐binding tag molecule ( i.e. a dinuclear zinc(II) complex) attached on a highly cross‐linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH 3 COONa, was necessary in the binding and washing buffers (0.10 M Tris‐CH 3 COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris‐CH 3 COOH, 10 mM NaH 2 PO 4 ‐NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate‐affinity chromatography using an epidermal growth factor‐stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS‐PAGE followed by Western blotting analysis.