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AIM:To prepare and characterize polyclonal antibodiesagainst aldose reductase-like (ARL-1) protein.METHODS:ARL-1 gene was inserted into bhe E.coliexpressionvector 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 fromdomestic rabbits immunized with ARL-(His)_6 were purifiedby CNBr-activated sepharose 4B coupled ARL-GST.Polyclonalantibodies were detected by Western blotting.RESULTS:Recombinant proteins of ARL-(His)_6 withmolecular weight of 35.7 KD and ARL-GST with molecularweight of 60.8 KD were highly expressed.The expressionlevels of ARL-GST and ARL-(His)_6 were 15.1% and 27.7%among total bacteria proteins,respectively.They weresoluble,predominantly in supernatant.After purification bynon-denatured way,SDS-PAGE showed one band.In thecourse of polyclonal antibodies purification,only one elutionpeak could be seen.Western blotting showed positive signalsin the two purified proteins and the bacteria transformedwith pGEX-4T-1(His)_6 C-ARL and pQE-30-ARL individually.CONCLUSION:Polyclonal antibodies are purified and highlyspecific against ARL-1 protein.ARL-GST and ARL-(His)_6 arehighly expressed and purified.
AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein. METHODS: ARL-1 gene was inserted into bhe 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.Polyclonalantibodies 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. Expression levels of ARL-GST and ARL- (His) _6 were 15.1% and 27.7 % among total bacteria proteins, respectively. They weresoluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. thecourse of polyclonal antibodies purification, only one elutionpeak could be seen. Western blotting showedpositivesin the two purified protei ns 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.