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| Production and Application of Polyclonal Antibody against p12, the Fourth Subunit of Human DNA Polymerase δ |
| LI Xiao, LIU Liu, ZHANG Lei, ZHOU Ya-jing |
| Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China |
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Abstract DNA polymerase δ (Pol δ) consists of p125, p68, p50 and p12 subunits. The smallest subunit p12 is thought to play a crucial and versatile role in modulating the functions of Pol δ during DNA replication and various DNA repair processes. In order to obtain polyclonal antibody against p12 with high specificity and sensitivity, the recombinant plasmid pGEX-5X-3-p12 was generated by PCR and subcloning of digested PCR product (p12 DNA) into the pGEX-5X-3. GST-tagged p12, expressed in BL21 cells which was transformed with pGEX-5X-3-p12 and induced by IPTG, was purified on Glutathione-Sepharose 4B beads and further purified on FPLC Mono Q column. Non-tagged p12, used as immunogen, was released by proteolysis with Factor Xa. The polyclonal antibody against p12 was produced by immunizing rabbits with highly purified non-tagged p12 protein. The collected antiserums were purified by precipitation with (NH4)2SO4 and followed by the purification on Protein A/G Plus Agarose. Western blotting analysis showed that the obtained antibody recognized endogenous p12 in good specificity and sensitivity. While a cell based immunocytochemistry assay showed a subcellular co-localization of p12 with PCNA to distinct nuclear spots which is the first time to confirm a physiological interaction of p12 with PCNA in vivo. Thus, the study provides a powerful tool for the further research on the p12 modulating Pol δ functions and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.
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Received: 31 January 2012
Published: 25 May 2012
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