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Preparation and characterization of polyclonal antibodies against ARL-1 protein
Jun-Fei Jin1, Liu-Di Yuan, Li Liu
1Genetics Research Center, Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China. wei.xie@seu.edu.cn
Aim:
To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein.
Methods:
ARL-1 gene was inserted into the E. coli expression vector pGEX-4T-1(His)(6)C and vector pQE-30. Recombinant ARL-1 proteins named ARL-(His)(6) and ARL-GST were expressed. They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His) (6) were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting.
Results:
Recombinant proteins of ARL-(His)(6) with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)(6) were 15.1 % and 27.7 % among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His) (6) C-ARL and pQE-30-ARL individually.
Conclusion:
Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His) (6) are highly expressed and purified.