Active Aldehyde Dehydrogenase 1 Family, Member A1 (ALDH1A1) (APE824Hu01)

ALDH1A1 (Retinal dehydrogenase 1) which belongs to the aldehyde dehydrogenase family, is the next enzyme after alcohol dehydrogenase in the major pathway of alcohol metabolism. ALDH1A1 converts retinaldehyde to retinoic acid. IL21 (interleukin 21) was identified as an interactor of ALDH1A1 through Affinity Capture-MS. Thus a binding ELISA assay was conducted to detect the interaction of recombinant human ALDH1A1 and recombinant human IL21. Briefly, ALDH1A1 were diluted serially in PBS, with 0.01%BSA (pH 7.4). Duplicate samples of 100uL ALDH1A1 were then transferred to IL21-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ALDH1A1 pAb, 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 ALDH1A1 and IL21 was shown in Figure 1, and this effect was in a dose dependent manner.

The activity of recombinant human ALDH1A1 was also measured by its ability to produce NADH during the oxidation of propionaldehyde. The reaction was performed in 0.1 M Tris, 0.1 M KCl, 2mM DTT, pH 8.5 (assay buffer), initiated by addition 50 μL of various concentrations of ALDH1A1 (diluted by assay buffer) to 50 μL of substrate mixture 2 mM beta-NAD and 20 mM propionaldehyde. The final well serves as a negative control with no ALDH1A1, replaced with 50 μl assay buffer. Read at a wavelength of 340 nm in kinetic mode for 5 minutes. The specific activity of recombinant human ALDH1A1 is > 130 pmol/min/μg.

Figure. SDS-PAGE