Active Cytochrome P450 1A2 (CYP1A2)

CYP1A2 (Cytochrome P450 1A2) belongs to the group of proteins which contains heme as a cofactor. CYP1A2 oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. Besides, ASAH1 (Acid ceramidase) has been identified as an interactor of CYP1A2 through affinity capture-MS. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human CYP1A2 and recombinant human ASAH1. Briefly, CYP1A2 were diluted serially in PBS, with 0.01%BSA (pH 7.4). Duplicate samples of 100uL were then transferred to ASAH1-coated microtiter wells and incubated for 2h at 37°C. Wells were washed with PBST and incubated for 1h with anti-CYP1A2 mAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of CYP1A2 and ASAH1 was shown in Figure 1, and this effect was in a dose dependent manner.