PHD finger protein 8蛋白多克隆抗体的制备、纯化与检测

Preparation, purification and identification of the polyclonal antibody of PHD finger protein 8

摘要:

PHD finger8 (PHF8) 蛋白是最新发现的一种带有PHD结构域和Jmjc结构域的蛋白。现有研究表明其可能在基因转录、组蛋白去甲基化等方面发挥重要作用。为研究其功能，本研究构建原核表达载体pET41b-PHF8 (aa886-936)，在大肠杆菌Escherichia coli BL21中诱导表达带有GST标签的PHF8 (aa886-936) 亲水片段融合蛋白，并纯化该片段作为抗原免疫家兔，再以CNBr活化Sepharose 4B微珠纯化抗血清制备PHF8特异性多克隆抗体。Western blotting以及免疫荧光检测表明该抗体具有很好的特异性，同时免疫荧光染色的结果也表明PHF8蛋白定位于细胞核。

Abstract:

PHD finger protein 8 (PHF8) is a novel protein with PHD domain and Jmjc domain, which may play important role in regulating transcription and histone demethylation. It is necessary to generate the antibody against PHF8 in order to further study its biological function. First we constructed plasmid pET41b-PHF8 (aa886-936) and expressed the GST-PHF8 (aa886-936) fusion protein in Escherichia coli BL21. We then purified the fusion protein by Glutathione Sepharose 4B beads and subjected to immunize the rabbits for acquiring antiserum. We obtained PHF8 polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. The antibody was effective in Western blotting and immunofluorescence with high specificity. Immunofluorescence also showed that PHF8 protein was located in nucleus in HeLa cells.