Polyclonal Antibody Preparation and Characterization of Mouse Rnf141

Objective To obtain the Rnf141 polyclonal antibody and to study its function. Methods Rnf141 cDNA was obtained by PCR amplification and inserted into the expression vector pGEX-5X-3, which encoding the GST protein to generate a recombinant plasmid. The recombinant was transformed into E.coli BL21(DE3). After inducing with 1 mmol/L IPTG at 25 ℃, the GST-Rnf141 fusion protein was expressed at a high level as a soluble form. Purified fusion protein with Glutathione Sephorase 4B chromatography was applied to immune rabbits to prepare the polyclonal antibody. Western blot and immunohistochemistry were applied to confirm the specificity of the resulting antibody. Results pGEX-Rnf141 recombinant plasmid was constructed successfully and the resulting. GST-Rnf141 fusion protein could be expressed in E.coli BL21(DE3) at a relatively high level, standing for 46% of total bacteria protein. The titer of the antiserum was 1∶128000 and the polyclonal antibody could recognize GST-Rnf141 fusion protein and Rnf141 protein in different tissues of mouse. The high expression of Rnf141 protein was observed in spleen and brain of mouse. Conclusion Rnf141 polyclonal antibody was prepared successfully and this will provide material for further studies of Rnf141 expression and function.