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Preparation and characterization of homeobox B9（HOXB9） polyclonal antibody
NIU Miao-miao MA Bo TANG Yan LI Feng ZHAN Jun* ZHANG Hong-quan
Acta Anatomica Sinica ›› 2013, Vol. 44 ›› Issue (4) : 468-474.
Preparation and characterization of homeobox B9（HOXB9） polyclonal antibody
Objective The purpose of this study was to prepare rabbit anti-homeobox B9(HOXB9) polyclonal antibody and to provide a tool for future study. Methods DNA fragments of human HOXB9 gene 5,terminal 7-458 base pairs was respectively cloned into PGEX-4T-1 vector expressing GST tag and pMal-C2x vector expressing MBP tag by polymerasechain reaction and recombinant DNA technology.The GST-HOXB9-N-terminal fusion protein and MBP-HOXB9-N-terminal fusion protein were expressed by E.coli BL21（DE3）after heat shock transformation and IPTG induction. The New Zealand rabbit was immunized with the purified GST-HOXB9-N-terminal fusion protein. We used ELISA assay to determine the titer of the antiserum.If the antiserum was effective, the rabbit blood was obtained from the carotid artery and immunoglobulinG(IgG)was purified from serum. Effectiveness and specificity of the antibody were identified by Western blotting and immunoprecipitation. Results The prepared antibody specifically recognized a single band of the target molecule, and had a strong capability of immunoprecipitation on the native HOXB9 protein.Using this antibody, we demonstrated that HOXB9 expressed highly in the mouse immune organs,pancreas,colon, lung, cerebellum, female reproductive system and testis,while expressed lowly in other organs. Conclusion We successfully prepared a rabbit anti-human HOXB9 polyclonal antibody. This polyclonal antibody is specific and effective, and may be used for Western blotting and immunoprecipitation analysis.
Homeobox B9 / Fusion protein / Polyclonal antibody / Western blotting / New Zealand rabbit
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