Recombinant MDA5 Enzyme

Raw polarization signal (mP) is converted to ADP formed using a a standard curve. Under initial velocity conditions, we recommend selecting an enzyme concentration that produces a 50-80% change in signal, corresponding to the linear phase of the reaction after conversion of values to ADP formation. Here, we use 100 µM ATP with 10 ng/µL dsRNA substrate. The enzyme buffer included 50 mM Tris, 2.5 mM MgCl₂, 0.01% Brij-35, 1.25 mM MnCl₂, and 5 mM DTT. The enzyme reaction took place for 120 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP² Alexa Fluor 633 Tracer, and 82 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.