Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes
Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes
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DOI：
10.1002/elps.200800386
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发表时间：
2009-02-01
期刊：
影响因子：
2.9
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Kinoshita, Eiji;Kinoshita-Kikutal, Emiko;Koike, Tohru
在此，我们描述了三种使用磷酸亲和电泳法分析磷蛋白亚型的2-DE。第一个维度是广泛使用的尿素-PAGE、IEF/NEPHGE或SDS-PAGE。第二个维度是使用磷酸盐结合标签分子PHOS-TAG(Mn2+-PHOS-TAG SDS-PAGE)的磷酸盐亲和SDS-PAGE。首次将尿素-PAGE和Mn2+-PHOS-TAG-SDS-PAGE联用，用于β-酪蛋白磷酸型的分离。用碱性磷酸酶部分去磷酸化制备了含有多种β-酪蛋白(有5个磷酸化位点)的磷酸异构体的典型蛋白质样品。将IEF/NEPHGE和Mn2+-PHOS-Tag-SDS-PAGE联用，对酪蛋白的磷酸型和Tau的体外激酶反应产物进行了分离。第三步将正常的SDS-PAGE和Mn2+-PHOS-Tag的SDS-PAGE联用，用于分离A431细胞在表皮生长因子刺激前后的裂解产物。随后用抗丝裂原活化蛋白激酶(MAPK)和抗Shc抗体进行免疫印迹，证实了刺激后MAPK1/2(44 kDa和42 kDa)和Shc(66 kDa、52 kDa和46 kDa)各蛋白亚型的磷酸化类型。通过这些新的二维方法，相对于目前的凝胶电泳法，包括一维Mn2+-PHOS-Tag-SDS-PAGE，磷酸蛋白亚型的分离应该得到改进。
Herein, we describe three kinds of 2-DE using phosphate-affinity PAGE for the analysis of phosphoprotein isotypes. The first dimension is a urea-PAGE, IEF/NEPHGE, or SDS-PAGE, which are widely used. The second dimension is a phosphate-affinity SDS-PAGE using a phosphate-binding tag molecule, Phos-tag (Mn2+-Phos-tag SDS-PAGE). The first 2-D procedure coupling urea-PAGE and Mn2+-Phos-tag SDS-PAGE was applied to the separation of beta-casein phosphoisotypes. A typical protein sample containing multiple phosphoisotypes from beta-casein (with five phosphorylation sites) was prepared by partial dephosphorylation with alkaline phosphatase. The second procedure coupling IEF/NEPHGE and Mn2+-Phos-tag SDS-PAGE was applied to the separations of phosphoisotypes of caseins and in vitro kinase reaction products of Tau. The third procedure coupling normal SDS-PAGE and Mn2+-Phos-tag SDS-PAGE was applied to the separation of A431 cell lysates before and after stimulation with an epidermal growth factor. This procedure followed by immunoblotting with anti-mitogen-activated protein kinase(MAPK) and anti-Shc antibodies demonstrated the detection of phosphoisotypes in each protein isoform of MAPK1/2 (44 and 42 kDa) and Shc (66, 52, and 46 kDa) after the stimulation. By these novel 2-D procedures, the separations of phosphoprotein isotypes should be improved relative to those by current gel electrophoresis methods, including 1-D Mn2+-Phos-tag SDS-PAGE.