A new approach to monitor expression of aldo-keto reductase proteins in mouse tissues

The aldo-keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan-AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan-AKR antibodies exhibited the immune specificities and immune sensitivities, Pan-AKR-P1 for AKR1B and AKR1C, Pan-AKR-P3 for AKR1C and Pan-AKR-P4 for all the AKR proteins. Pan-AKR-P4 antibody was employed to 2-DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune-reactive spots from each tissue. Protein identification with MS revealed that most immune-positive spots were the members of AKR superfamily. Furthermore, Pan-AKR-P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.