Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH
Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH
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DOI：
10.1002/pmic.200600252
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发表时间：
2006-10-01
期刊：
影响因子：
3.4
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Kinoshita-Kikuta, Emiko;Kinoshita, Eiji;Koike, Tohru
尽管磷蛋白在后基因组研究（如临床诊断和药物设计）中引起了极大的兴趣，但从细胞或组织中特异性富集天然磷蛋白的方法却很少。在这里，我们描述了一个简单而有效的协议，以丰富磷蛋白全面从一个复杂的混合物含有溶解的细胞蛋白。该方法是基于固定化金属亲和色谱法，其使用附着在高度交联琼脂糖上的磷酸盐结合标签分子（即双核锌（II）络合物）。结合、洗涤和洗脱过程均在pH 7.5和室温下在没有去污剂或还原剂的情况下进行。在结合和洗涤缓冲液（0.10 M Tris-CH 3COOH，pH 7.5）中需要添加剂1.0 M CH 3COONa，以防止非磷酸化蛋白结合。使用由0.10 M Tris-CH 3COOH、10 mM NaH 2 PO 4-NaOH和1.0 M NaCl组成的混合缓冲溶液（pH 7.5）洗脱吸收的磷蛋白。在这项研究中，我们展示了一个典型的例子，磷酸盐亲和层析使用表皮生长因子刺激的A431细胞裂解物。柱层析（1 mL凝胶规模）的总时间小于1 h。使用SDS-PAGE随后进行Western印迹分析来评估磷蛋白到洗脱级分中的强富集。
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.