Preparation and detection of centromere-associated protein E antibody / 国际生物医学工程杂志

ABSTRACT

Objective To prepare centromere-associated protein E(CENP-E)polyclonal antibody with specificity by using New Zealand white rabbits.Methods Prokaryotic expression plasmid of pHis-CENPEC410 was constructed by molecular cloning technique and then transformed into competent cells of E.coli BL-21 (DE3).HisCENPEC410 fusion protein was induced by isopropyl β-D-thiogalactoside (IPTG) and purified by affinity chromatography using Ni-NTA beads.The purified protein was used as antigen to immune New Zealand white rabbits to produce spccific polyclonal antibody of CENP-E.The antibodies serum was detected by immunoblotting and co-immunoprecipitation,and the purified antibodies were detected by immunofluorescene staining.Results The results of immunoblotting and co-immunoprecipitation demonstrated that the antibody serum was effective and the purified antibody could be applied to immunofluorescene test.Conclusions CENP-E polyclonal antibody with high specificity and sensitivity was obtained,which lay the foundation for the follow-up study of CENP-E.