[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 degrees C, 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.


CONCLUSIONS
Rnf141 polyclonal antibody was prepared successfully and this will provide material for further studies of Rnf141 expression and function.
Song
Hong-xia
HX
Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhang
Si-zhong
SZ
Yang
Ji-yun
JY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C486693:Rnf141 protein, mouse; D014157:Transcription Factors