Yim Sun Hee, Tobe Ryuta, Turanov Anton A, Carlson Bradley A
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
College of Life Sciences, Ritsumeikan University, Kusatsu, Japan.
Methods Mol Biol. 2018;1661:177-192. doi: 10.1007/978-1-4939-7258-6_13.
The trace element selenium (Se) is incorporated into proteins as the amino acid selenocysteine (Sec), which is cotranslationally inserted into specific proteins in response to a UGA codon. Proteins containing Sec at these specific positions are called selenoproteins. Most selenoproteins function as oxidoreductases, while some serve other important functions. There are 25 known selenoprotein genes in humans and 24 in mice. The use of Sec allows selenoproteins to be detected by a convenient method involving metabolic labeling with Se. Labeling of cells and whole animals are used for the examination of selenoprotein expression profiles and the investigation of selenoprotein functions. In mammals, nonspecific Se insertion is very low, and sensitivity and specificity of selenoprotein detection approaches that of Western blotting. This method allows for the examination of selenoprotein expression and Se metabolism in model and non-model organisms. Herein, we describe experimental protocols for analyzing selenoproteins by metabolic labeling with Se both in vitro and in vivo. As an example, the procedure for metabolic labeling of HEK293T human embryonic kidney cells is described in detail. This approach remains a method of choice for the detection of selenoproteins in diverse settings.
微量元素硒（Se）作为氨基酸硒代半胱氨酸（Sec）掺入蛋白质中，Sec会响应UGA密码子而共翻译插入特定蛋白质中。在这些特定位置含有Sec的蛋白质称为硒蛋白。大多数硒蛋白发挥氧化还原酶的功能，而有些则具有其他重要功能。人类已知有25个硒蛋白基因，小鼠有24个。Sec的使用使得硒蛋白能够通过一种便捷的方法进行检测，该方法涉及用硒进行代谢标记。细胞和全动物的标记用于检查硒蛋白表达谱以及研究硒蛋白功能。在哺乳动物中，非特异性硒插入非常低，硒蛋白检测的灵敏度和特异性与蛋白质免疫印迹法相当。该方法可用于检查模式生物和非模式生物中的硒蛋白表达及硒代谢。在此，我们描述了通过体外和体内用硒进行代谢标记来分析硒蛋白的实验方案。作为示例，详细描述了HEK293T人胚肾细胞的代谢标记程序。这种方法仍然是在各种情况下检测硒蛋白的首选方法。
