Kinoshita Eiji, Kinoshita-Kikuta Emiko, Koike Tohru
Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8553, Japan.
Methods Mol Biol. 2017;1599:113-126. doi: 10.1007/978-1-4939-6955-5_9.
In this chapter, we provide a standard protocol for phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zn-Phos-tag SDS-PAGE). This technique uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system, Tris [tris(hydroxymethyl)aminomethane], and acetic acid (Tris-AcOH), to detect shifts in the mobility of phosphorylated ataxia telangiectasia-mutated (ATM) kinase. This protocol, which employs a 3% (w/v) polyacrylamide gel strengthened with 0.5% (w/v) agarose, permits the separation of larger phosphoproteins with molecular masses in the order of 200 kDa over a period of approximately 4 h. Subsequently, multiple phosphorylated forms of high-molecular-mass ATM kinase (350 kDa) can be clearly detected via immunoblotting as multiple upshifted migration bands on the Zn-Phos-tag SDS-PAGE gel. The procedure described in this protocol requires a completion time of approximately 5 h from the beginning of gel preparation to the end of electrophoresis.
在本章中，我们提供了一种用于磷酸亲和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（锌 - 磷酸标签SDS - PAGE）的标准方案。该技术使用磷酸结合分子磷酸标签的二锌（II）配合物，结合中性pH凝胶系统、三（羟甲基）氨基甲烷（Tris）和乙酸（Tris - AcOH），来检测磷酸化的共济失调毛细血管扩张突变（ATM）激酶迁移率的变化。该方案采用用0.5%（w/v）琼脂糖增强的3%（w/v）聚丙烯酰胺凝胶，在约4小时内可分离分子量约为200 kDa的较大磷酸化蛋白。随后，通过免疫印迹法可以在锌 - 磷酸标签SDS - PAGE凝胶上清晰地检测到高分子量ATM激酶（350 kDa）的多种磷酸化形式，表现为多个上移的迁移条带。本方案中描述的程序从凝胶制备开始到电泳结束大约需要5小时完成。
