| Assay Method Information | |
| | ALDH Assay Protocol |
| Description: | 5 μl of enzyme (150 nM for ALDH1a1 and 200 nM for ALDH2 in reaction buffer) were delivered to assay wells in coming black, 384 well plate. 5 μl of reaction buffer was delivered to ‘no enzyme’ background control wells. Test compounds were prepared in 100% DMSO by serial dilution in 100× of assay concentration. After adding the test compounds, reaction plate was centrifuged briefly in 1200 rpm, then incubated for 20 min at room temperature, to pre-incubate enzyme and compounds. 5 μl of substrate solution (reaction buffer containing 250 μM Acetaldehyde, 500 μM NAD+ for ALDH1a1 and 100 μM Acetaldehyde, 500 μM NAD+ in the for ALDH2) were then delivered to assay wells. Reaction plate was briefly centrifuged and sealed with a plastic film to limit evaporation. After incubation at room temperature for 60 min, 10 μl of detection reagent (15 μg/ml diaphorase, 30 μM resazurin prepared in the degassed reaction buffer) was added. Reaction plate was briefly centrifuged and incubated for 10 minutes at room temperature in the dark. Fluorescent signal from resorufin was measured by Perkin Elmer Envision at Ex/Em=535/590 nm. |
| Affinity data for this assay | |
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