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| Preparation and Preliminary Application of Human Protein DFF45 Polyclonal Antibody |
| Tianjin Life Science Research Center Basic Medical School of Tianjin Medical University， Tianjin 300070， China |
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Abstract To express and purify the fusion protein of GST-tagged DNA fragmentation factor 45(DFF45) in prokaryocytes and prepare anti-DFF45 polyclonal antibody. The expression plasmid pGEX-5X-1/DFF45 was constructed and transformed into E.coli BL21, and the expression of GST-DFF45 protein was induced by IPTG. After purification, the fusion protein GST-DFF45 was used to immunize Oryctolagus cuniculus to obtain the antibody serum. Then the anti-DFF45 serum was purified by CNBr-activated Sepharose? 4B. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blotting and immunofluorescence. Furthermore, we detected the expression of DFF45 protein in several human cell lines, and observed the cellular localization of DFF45 protein. The fusion protein was successfully expressed and purified, and the specific polyclonal antibody was prepared. The titer of the polyclonal antibody was up to 1:20 000 detected by ELISA, and the high specificity of the antibody was confirmed by Western blotting and immunofluorescence. Moreover, we found that DFF45 protein showed differential expression in several human cell lines. We also found that DFF45 protein was located in both cytoplasm and nucleus. The successful preparation of DFF45 polyclonal antibody and the confirmation of its different expression levels in human cells provides a tool for the further study of the relation between DFF45 and tumors.
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Received: 26 November 2009
Published: 26 February 2010
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