Liver Cancer
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1455-1459
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1455
Preparation and characterization of polyclonal antibodies against ARL-1 protein
Jun-Fei Jin, Liu-Di Yuan, Li Liu, Zhu-Jiang Zhao, Wei Xie
Jun-Fei Jin, Liu-Di Yuan, Li Liu, Zhu-Jiang Zhao, Wei Xie, Genetics Research Center, Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China
Author contributions: All authors contributed equally to the work.
Supported by Health Department Grant of Jiang Su Province (H9925) and National Award for Excellent Research and Teaching from the Ministry of Education (2001/182)
Correspondence to: Dr. Wei Xie, Genetics Research Center, Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China. wei.xie@seu.edu.cn
Telephone: +86-25-3220761 Fax: +86-25-3220761
Received: November 19, 2002
Revised: December 4, 2002
Accepted: December 18, 2002
Published online: July 15, 2003
Abstract

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)6C 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.

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