Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma, serum, as well as many other biological fluids
1. Standard Curve Preparations: For colorimetric assay, add 0, 4, 8, 12, 16, 20 µL into each well individually. Adjust volume to 50 µL/well with Uric Acid Assay Buffer to generate 0, 8, 16, 24, 32, 40 nM/well of Uric Acid Standard. For fluorometric assay, dilute the Uric Acid to 0.2 nM/µL by adding 20 µL into 180 µL of Uric Acid Assay Buffer. Mix well. Add 0, 4, 8, 12, 16, 20 µL into each well individually. Adjust volume to 50 µL/well with Uric Acid Assay Buffer to generate 0, 0.8, 1.6, 2.4, 3.2, 4.0 nM/well of the Uric Acid Standard.
2. Sample Preparations: Prepare test samples in 50 µL/well with Uric Acid Assay Buffer in a 96-well plate. If using serum sample, serum (2-20 µL/assay, normal serum contains approx. 0.3 nM/µL uric acid) can be directly diluted in the Uric Acid Assay Buffer. We suggest using several dilutions to ensure that the readings are within the standard curve range.
3. Reaction Mix Preparation: Mix enough reagents for the number of assays performed: For each well, prepare a total 50 µL Reaction Mix containing: 46 µL Uric Acid Assay Buffer 2 µL Uric Acid Probe 2 µL Uric Acid Enzyme Mix,
4. Mix well. Add 50 µL of the Reaction Mix to each well that contains the uric acid standard and test samples. Incubate the reaction for 30 minutes at 37 °C, protect from light.
5. Measure OD 570nm for colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a microplate reader.