1. Prepare all reagents, samples and standards;
2. Add 25uL standard or sample to each well. Incubate 1 hour at 37℃;
3. Aspirate and add 25uL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 25uL prepared Detection Reagent B. Incubate 30 minutes at 37℃;
6. Aspirate and wash 5 times;
7. Add 25uL Substrate Solution. Incubate 10-20 minutes at 37℃;
8. Add 20uL Stop Solution. Read at 450nm immediately.
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