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Amplite® Fluorimetric Ascorbic Acid Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples with diluted ascorbic acid standards (50 µL)
- Add equal volume of working solution (50 µL)
- Incubate at room temperature for 30 minutes to 1 hour
- Monitor fluorescence intensity at Ex/Em = 340/430 nm
Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. Ascorbrite™ Blue stock solution (200X):
Add 50 µL of DMSO (Component E) into Ascorbrite™ Blue (Component A) to make 200X Ascorbrite™ Blue stock solution.
2. Ascorbic Acid standard solution (100 mM):
Add 200 µL of ddH2O into ascorbic acid standard vial (Component D) to make 100 mM ascorbic acid standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13835
Add 10 µL of 100 mM ascorbic acid into 990 µL of assay buffer to get 1000 µM ascorbic acid solution (AA7). Then take the 1000 µM ascorbic acid standard solution and perform 1:3 serial dilutions to get serially diluted ascorbic acid standards (AA1 - AA6). Note: Ascorbic acid aqueous solution is not stable and will oxidize into DHA.
PREPARATION OF WORKING SOLUTION
Total AA Assay
Add 5 mL of Assay Buffer (Component C) into one bottle of Enzyme Mix (Component B). Then add 25 uL of AscorbriteTM Blue stock Solution (200X) into the same bottle. Mix well.
DHA Assay
In an appropriate container, add 5 mL of Assay Buffer (Component C) with 25 uL of AscorbriteTM Blue stock Solution (200X). Mix well. Note: Alternatively, one can make enzyme stock solution by adding 100 μL ddH2O into one Enzyme Mix bottle (Component B) to make 50X enzyme stock solution, and use it proportionally for total AA assay (for example, for 1mL total AA assay working solution, add 20 μL 50X enzyme stock solution and 5 μL 200X Ascorbrite™ Blue stock Solution into 1 mL Assay Buffer). Note: The working solution is not stable. Use promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of ascorbic acid standards and test samples in a solid black 96-well microplate. AA = Ascorbic Acid Standard (AA1-AA7, 1 to 1000 µM); BL = blank control; TS = test sample.
| BL | BL | TS | TS |
| AA1 | AA1 | ... | ... |
| AA2 | AA2 | ... | ... |
| AA3 | AA3 | ||
| AA4 | AA4 | ||
| AA5 | AA5 | ||
| AA6 | AA6 | ||
| AA7 | AA7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| AA1-AA7 | 50 µL | Serial Dilution (1 to 1000 µM) |
| BL | 50 µL | Assay Buffer (Component C) |
| TS | 50 µL | Test Sample |
- Prepare ascorbic acid standards (AA), blank control (BL), and test samples (TS) according to the layout of Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of working solution into each well of ascorbic acid standard, blank control, and test samples to make the total ascorbic acid assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 30 minutes to 1 hour.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 340/430 nm (cut off: 420 nm).
References
Authors: Abad S, Perez X, Planas A, Turon X.
Journal: Talanta (2014): 210
Authors: Lowe HI, Toyang NJ, Watson CT, Bryant J.
Journal: Nat Prod Commun (2014): 687
Authors: Rohe A, Gollner C, Erdmann F, Sippl W, Schmidt M.
Journal: J Enzyme Inhib Med Chem (2014): 514
Authors: Willias SP, Chauhan S, Motin VL.
Journal: Microb Pathog (2014): 33
Authors: Dong Y, Ma Y, Yan K, Shen L, Wang X, Xu Y, He G, Wu Y, Lu J, Yang Z, Feng F.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2014): 30
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