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The antibody titer and specificity were determined by ELISA and western blot analysis, respectively. The tissue distribution of pSix1 was determined by western blot using the prepared polyclonal antibody. | Turkish Journal of Biology http://journals.tubitak.gov.tr/biology/ Research Article Turk J Biol (2015) 39: 335-342 © TÜBİTAK doi:10.3906/biy-1408-66 Prokaryotic expression, purification, polyclonal antibody preparation, and tissue distribution of porcine Six1 Meng XU*, Xiaoling CHEN*, Zhiqing HUANG**, Wanxue WEN, Shuai CHANG, Xiaoyan WANG, Daiwen CHEN, Bing YU, Junqiu LUO, Guangmang LIU Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan, P. R. China Received: 27.08.2014 Accepted: 22.11.2014 Published Online: 01.04.2015 Printed: 30.04.2015 Abstract: Sine oculis homeobox 1 (Six1), a member of the Six homeoproteins, plays an important role in skeletal myogenesis and the specification of myofiber diversity. In this study, in order to scale up the production of recombinant porcine Six1 (pSix1), a pET-30a(+)pSix1 plasmid was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant pSix1 could be induced for efficient expression with 2 mM IPTG for 2 h at 30 °C, yielding approximately 4.6 mg/L. The protein was then purified and identified by western blot, and it was used for preparing its polyclonal antibody. The recombinant pSix1 was tagged with a 6-His tag at its C-terminus, which could be conveniently purified by affinity column. The purified recombinant protein was used for immunizing Sprague Dawley rats to obtain the polyclonal antibody against pSix1. The antibody titer and specificity were determined by ELISA and western blot analysis, respectively. The tissue distribution of pSix1 was determined by western blot using the prepared polyclonal antibody. Key words: Porcine Six1, Escherichia coli, expression, purification, identification, polyclonal antibody, tissue distribution 1. Introduction Pigs are raised throughout the world as a meat production animal. Selection for high growth rate and lean meat deposition has resulted
