Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.
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 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 mixed 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 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.
Kinoshita-Kikuta
Emiko
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Kinoshita
Eiji
E
Yamada
Atsushi
A
Endo
Mika
M
Koike
Tohru
T
D002457:Cell Extracts / Q000737:chemistry*; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation
D002457:Cell Extracts; D010750:Phosphoproteins