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Protokoll
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1. Standard Curve Preparations: For the colorimetric assay, dilute the standard to 1 mM by adding 10 μl of the 100 mM Ascorbic Acid Standard to 990 μl of distilled water, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 120 μl/well with Ascorbic Acid Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Ascorbic Acid Standard. For the fluorometric assay, dilute the Ascorbic Acid Standard to 0.01- 0.1 mM with the Ascorbic Acid Assay Buffer (Note: Detection sensitivity is 10 to 100 fold higher for a fluorometric than a colorimetric assay). Follow the procedure for the colorimetric assay. Note: Diluted ascorbic acid standard is unstable, use fresh dilution each time. 2. Sample Preparation: Prepare test samples to a final volume of 120 μl/well with Ascorbic Acid Assay Buffer in a 96-well plate. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. NOTE: 1) Due to high protein contents and other compounds present in serum we recommend using FRASC Ascorbic Acid Kit (K671-100) for serum samples. 2) Ascorbate is easily oxidized during sample preparation and great care must be exercised to achieve quantitative recovery. 3. Catalyst: Add 100 μl of catalyst to 900 μl of distilled water and vortex well. 4. Add 30 μl of catalyst to each standard and sample well. 5. Ascorbic Acid Reaction Mix: Mix enough reagents for the number of samples and standards to be performed: For each well, prepare a total 50 μl Reaction Mix containing: 46 μl Ascorbic Acid Assay Buffer 2 μl Ascorbic Acid Probe 2 μl Ascorbic Acid Enzyme Mix 6. Mix well. Add 50 μl of the Reaction Mix to each well containing the Ascorbic Acid Standard and test samples. Mix well. 7. Protect from light, Color is developed within 3 minutes and stable for an hour. 8. Measure O.D. 570nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay in a micro-plate reader. 9. Correct background by subtracting the value derived from the 0 ascorbic acid standard from all sample readings (Note: The background reading can be significant and must be subtracted from sample readings). Apply sample readings to the generated standard curve. Ascorbic Acid concentration can then be calculated: C = As / Sv nmol/ μl or μ mol/ml or mM Where: As is ascorbic acid amount from standard curve (nmol). Sv is the sample volume added in sample wells ( μl). Ascorbic Acid molecular weight: 176.12. VI.
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