Specificity and application of SOX2 antibody.
Specificity and application of SOX2 antibody.
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SOX2抗体的特异性及应用
DOI：
10.1016/j.psj.2020.01.001
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发表时间：
2020-05
期刊：
影响因子：
4.4
通讯作者：
Zheng X
中科院分区：
文献类型：
--
作者：
Wei L;Wang L;Pan J;Liu L;Wang P;Wei J;Xu X;Xing Q;Liu P;Wu W;Li G;Zheng X
Sox 2在维持胚胎干细胞的全能性和自我更新中起重要作用。本研究的目的是利用原核表达技术制备抗鸡Sox 2多克隆抗体，并对其特异性进行评价，同时研究Sox 2在鸡脑和肺组织中的表达和分布。扩增鸡Sox 2基因，亚克隆至pET-30 a载体，构建原核表达载体pET-Sox 2。表达、纯化His-Sox 2融合蛋白，并用于制备抗鸡Sox 2多克隆抗体。Western blotting结果表明，抗鸡Sox 2抗体不仅能与纯化的His-Sox 2融合蛋白结合，而且能与鸡睾丸内源性Sox 2蛋白结合，并呈现明显的剂量依赖关系。Sox 2过表达细胞的间接免疫荧光染色显示，在抗鸡Sox 2抗体染色细胞中，Sox 2在细胞核和细胞质中呈强免疫反应性。CRISPR/Cas9效应系统介导的Sox 2敲低测定表明，HEK 293 T细胞中的Sox 2表达在多西环素存在下下调，但在多西环素不存在下上调。此外，冷冻切片和免疫组化染色表明，大多数精原细胞的曲细精管，和少数支持细胞和Leydig细胞，是阳性的Sox 2。抗鸡Sox 2抗体也被成功地用于研究Sox 2在鸡小脑皮质，视顶盖，大脑皮质和肺中的表达和分布。本研究结果证实了抗鸡Sox 2多克隆抗体的特异性，为研究鸡Sox 2基因的生物学功能和鸡多能干细胞的自我更新机制奠定了基础。
Sox2 is known to play an important role in maintaining the totipotency and self-renewal of embryonic stem cells. The purpose of this study was to prepare an anti-chicken Sox2 polyclonal antibody using prokaryotic expression techniques, to evaluate its specificity and to use it to investigate the expression and distribution of Sox2 in the chicken brain and lungs. The chicken Sox2 gene was amplified and subcloned to a pET-30a vector to construct a prokaryotic expression vector, pET-Sox2. A His-Sox2 fusion protein was expressed, purified, and used to prepare an antichicken Sox2 polyclonal antibody. Western blotting revealed that the antichicken Sox2 antibody could specifically bind not only to the purified His-Sox2 fusion protein but also to the endogenous Sox2 protein in the testes of chicken, showing a distinct dose-dependent relationship between antigen and Sox2 antibody. Indirect immunofluorescent staining of Sox2-overexpressing cells showed strong nuclear and diffuse cytoplasmic immunoreactivity for Sox2 in the antichicken Sox2 antibody-staining cells. A CRISPR/Cas9 effector system-mediated Sox2 knockdown assay indicated that Sox2 expression in HEK 293T cells was downregulated in the presence of doxycycline but upregulated in the absence of doxycycline. In addition, cryosectioning and immunohistochemical staining illustrated that most spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken Sox2 gene and the self-renewal mechanisms of chicken pluripotent stem cells.
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影响因子：
3
作者：
Shitasako, Shiori;Ito, Yoko;Ohshima, Toshio
通讯作者：
Ohshima, Toshio
影响因子：
3.7
作者：
Tompkins DH;Besnard V;Lange AW;Wert SE;Keiser AR;Smith AN;Lang R;Whitsett JA
通讯作者：
Whitsett JA
影响因子：
46.9
作者：
Danwei Huangfu;Osafune, Kenji;Melton, Douglas A.
通讯作者：
Melton, Douglas A.
影响因子：
--
作者：
Nettersheim D;Heimsoeth A;Jostes S;Schneider S;Fellermeyer M;Hofmann A;Schorle H
通讯作者：
Schorle H
影响因子：
9.9
作者：
Nielsen AA;Voigt CA
通讯作者：
Voigt CA