| 睾丸特异表达蛋白质RNF138的表达纯化及抗体制备 |
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| 引用本文： | 梁俊波,程筱雯,缪时英,王琳芳.睾丸特异表达蛋白质RNF138的表达纯化及抗体制备[J].河南师范大学学报(自然科学版),2011,39(3):111-114,166. |
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| 作者姓名： | 梁俊波 程筱雯 缪时英 王琳芳 |
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| 作者单位： | 中国医学科学院,基础医学研究所,医学分子生物学国家重点实验室,北京协和医学院基础学院,北京100005 |
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| 基金项目： | 国家重大科学研究计划项目 |
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| 摘 要： | 目的:制备RNF138的多克隆抗体,为进一步研究其功能奠定基础.方法:通过RT-PCR从小鼠睾丸cDNA文库中扩增mRNF138的开放阅读框,并克隆至原核表达载体pET-30a中;将测序正确的pET-30a(+)/mRNF138重组质粒转化大肠杆菌Rosseta菌株,经IPTG诱导表达后通过镍离子螯合柱(Ni-NTA)...
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| 关 键 词： | RNF138 基因克隆 蛋白表达纯化 Western Blot |
Expression, Purification of Testis Specifically Expressed Protein RNF138 and Antibody Preparation Against RNF138 |
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| LIANG Jun-bo,CHENG Xiao-wen,MIAO Shi-ying,WANG Lin-fang.Expression, Purification of Testis Specifically Expressed Protein RNF138 and Antibody Preparation Against RNF138[J].Journal of Henan Normal University(Natural Science),2011,39(3):111-114,166. |
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| Authors: | LIANG Jun-bo CHENG Xiao-wen MIAO Shi-ying WANG Lin-fang |
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| Institution: | (National Key Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,CAMS and PUMC,Beijing 100005,China) |
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| Abstract: | Objective To characterize the function of RNF138 in spermatogenesis,RNF138 polyclonal antibody is raised using mouse RNF138(mRNF138) recombinant protein as immunogen.MethodsmRNF138 open reading frame is amplified from mouse testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pET-30a(+).The recombinant plasmid verifying by sequencing is transformed into E.coli.Rosseta strain.The expression of pET-30a(+)/mRNF138 plasmid is induced by IPTG and the corresponding protein is purified by nickel-chelating column(Ni-NTA) and gel-cutting.mRNF138 polyclonal antibody is obtained by immunizing New Zealand white rabbits.Results ELISA results show that the titer of mRNF138 is up to 1∶32 million.Except for detecting recombinant mRNF138 protein,the antibody could also effectively identify endogenous RNF138 protein. |
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| Keywords: | RNF138 gene cloning protein expression and purification antibody Western Blot |
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