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Amplite® Rapid Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*
Example protocol
AT A GLANCE
Protocol summary
- Prepare GSH working solution (50 µL)
- Add GSH standards or test samples (50 µL)
- Incubate at RT for 10 to 60 minutes
- Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. GSH standard solution (1 mM):
Add 200 µL of ddH2O into the vial of GSH Standard (Component C) to make 1 mM (1 nmol/µL) GSH standard solution.
2. Thiolite™ Green 520WS stock solution (100X):
Add 100 µL of ddH2O into the vial of Thiolite™ Green 520WS (Component A) to make 100X Thiolite™ Green 520WS stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/5528
Add 30 μL of 1 mM (1 nmol/µL) GSH standard solution to 970 μL of Assay Buffer (Component B) to generate 30 μM (30 pmol/µL) GSH standard solution. Take 30 μM (30 pmol/µL) GSH standard solution and perform 1:3 serial dilutions to get serially diluted GSH standards (SD7-SD1) with Assay Buffer (Component B). Note: Diluted GSH standard solution is unstable. Use within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 50 μL of 100X Thiolite™ Green 520WS stock solution into 5 mL of Assay Buffer (Component B) and mix well to make GSH working solution. Note: This GSH working solution is enough for one 96-well plate. It is stable at 40C for 6 hours when kept from light. Note: Alternatively, one can make GSH working solution by adding 100X Thiolite™ Green 520WS stock solution with Assay Buffer (Component B) proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of GSH standards and test samples in a solid black 96-well microplate. SD = GSH Standards (SD1 - SD7, 0.014 to 10 µM); BL=Blank Control; TS=Test Samples
| BL | BL | TS | TS |
| SD1 | SD1 | ... | ... |
| SD2 | SD2 | ... | ... |
| SD3 | SD3 | ||
| SD4 | SD4 | ||
| SD5 | SD5 | ||
| SD6 | SD6 | ||
| SD7 | SD7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| SD1-SD7 | 50 µL | Serial Dilutions (0.014 to 10 µM) |
| BL | 50 µL | Assay Buffer |
| TS | 50 µL | Test Sample |
- Prepare GSH standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat cells or tissue samples as desired.
- Add 50 µL of GSH working solution to each well of GSH standard, blank control and test samples to make the total assay volume 100 µL/well. For a 384-well plate, add 25 µL of GSH working solution into each well instead, for total volume of 50 µL/well.
- Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).
Citations
Authors: Sekine, Takashi and Hirata, Tadashi and Mine, Toshiki and Fukano, Yasuo
Journal: Toxicology mechanisms and methods (2016): 22--31
References
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Journal: Anal Biochem (2007): 175
Authors: Gan J, Harper TW, Hsueh MM, Qu Q, Humphreys WG.
Journal: Chem Res Toxicol (2005): 896
Authors: Storhoff JJ, Marla SS, Bao P, Hagenow S, Mehta H, Lucas A, Garimella V, Patno T, Buckingham W, Cork W, Muller UR.
Journal: Biosens Bioelectron (2004): 875
Authors: Christiansen C, St Hilaire PM, Winther JR.
Journal: Anal Biochem (2004): 148
Authors: Bayle C, Issac C, Salvayre R, Couderc F, Causse E.
Journal: J Chromatogr A (2002): 255
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