Komis George, Takáč Tomáš, Bekešová Slávka, Vadovič Pavol, Samaj Jozef
Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, 783 71, Olomouc, Czech Republic,
Methods Mol Biol. 2014;1171:47-63. doi: 10.1007/978-1-4939-0922-3_5.
Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.
蛋白质磷酸化是最丰富且研究最深入的蛋白质翻译后修饰，致力于调节蛋白质功能、亚细胞定位以及蛋白质 - 蛋白质相互作用。动态、条件性蛋白质磷酸化的鉴定和定量可通过以下方法实现：用（32）P 标记的 ATP 对目标蛋白质进行代谢标记，随后进行放射自显影分析；使用针对磷酸化蛋白质种类的特异性单克隆或多克隆抗体；最后通过质谱法描绘磷酸化蛋白质组。在此，我们提出第四种方法，该方法依赖于在与 Phos - Tag™以及 Mn(2+)或 Zn(2+)阳离子共聚的标准 SDS - PAGE 系统中，基于亲和作用对磷酸化和非磷酸化蛋白质种类进行电泳分离。磷酸化的丝氨酸、苏氨酸和酪氨酸残基的磷酸基团与固定在聚丙烯酰胺上的 Phos - Tag™以及 Mn(2+)和 Zn(2+)阳离子形成复合物。对凝胶进行适当处理后，分离出的蛋白质可定量转移至 PVDF 或硝酸纤维素膜上，并用通用的非磷酸化状态特异性抗体进行检测，从而确定特定磷酸化蛋白质种类相对于其非磷酸化对应物的存在情况。
