Two-dimensional Phos-tag affinity electrophoresis

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  • Kimura Yayoi
    Graduate School of Nanobioscience, Yokohama City University
  • Nagata Kayoko
    Graduate School of Nanobioscience, Yokohama City University
  • Hirano Hisashi
    Graduate School of Nanobioscience, Yokohama City University Advanced Medical Research Center, Yokohama City University
  • Ohara Osamu
    Laboratory for Immunogenomics, RIKEN Research Center for Allergy and Immunology Kazusa DNA Research Institute

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Other Title
  • 二次元Ｐｈｏｓ－ｔａｇ親和性電気泳動
  • ニジゲン Phos-tag シンワセイ デンキ エイドウ

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Abstract

Two-dimensional electrophoresis using Phos-tag affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of protein. When we used 2-DE with isoelectric focusing using IPG gel strip in the first dimension and Phos-tag affinity electrophoresis in the second dimension, the nuclear fraction contained more than 20 spots of endogenous heterogeneous nuclear ribonucleoprotein K (hnRNP K) on the 2-DE map. Mass spectrometric analysis indicated that these multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. By applying this two-dimensional electrophoresis map of hnRNP K, we indicated that the multiple forms were differentially modulated in response to external stimulation.<br>

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