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Amplite® Colorimetric Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare G6PD working solution (50 µL)
- Add G6PD standards or test samples (50 µL)
- Incubate at room temperature for 30 minutes - 2 hours
- Monitor absorbance ratio increase at A575nm/A605nm
Important notes
Thaw each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NADP stock solution (100X):
Add 100 µL of H2O into the vial of NADP (Component C) to make 100X NADP stock solution.
2. G6PD standard solution (100 U/mL):
Add 100 µL of H2O or 1X PBS buffer into the vial of G6PD Standard (Component D) to make 100 U/mL G6PD standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13807
Add 10 µL of 100 U/mL G6PD standard solution into 990 µL 1x PBS buffer to generate 1000 mU/mL G6PD standard solution. Take 1000 mU/mL G6PD standard solution and perform 1:3 serial dilutions in 1x PBS buffer to get serially diluted G6PD standards (G6PD7 - G6PD1). Note: Diluted G6PD standard solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A), and mix well.
2. Add 50 µL of 100X NADP stock solution into the bottle of Component A+B and mix well to make G6PD working solution. Note: This G6PD working solution is enough for one 96-well plate. It is unstable at room temperature and should be used promptly within 2 hours. Avoid exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of G6PD standards and test samples in a white clear bottom 96-well microplate. G6PD=D-Glucose-6-Phosphate Dehydrogenase Standards (G6PD1 - G6PD7, 0.4 to 300 mU/mL), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| G6PD1 | G6PD1 | ... | ... |
| G6PD2 | G6PD2 | ... | ... |
| G6PD3 | G6PD3 | ||
| G6PD4 | G6PD4 | ||
| G6PD5 | G6PD5 | ||
| G6PD6 | G6PD6 | ||
| G6PD7 | G6PD7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| G6PD1 - G6PD7 | 50 µL | Serial Dilutions (0.4 to 300 mU/mL) |
| BL | 50 µL | Dilution Buffer |
| TS | 50 µL | test sample |
- Prepare G6PD standards (G6PD), 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.
- Add 50 µL of G6PD working solution to each well of G6PD standard, blank control, and test samples to make the total G6PD assay volume of 100 µL/well. For a 384-well plate, add 25 µL of G6PD working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
- Monitor the absorbance ratio increase with an absorbance plate reader at A575nm/A605nm.
Citations
Authors: Tanaka, Satoshi and Akagawa, Hiroyuki and Azuma, Kenkou and Watanabe, Kaoru and Higuchi, Sayaka and Iwasaki, Naoko
Journal: Endocrine Journal (2023): EJ22--0541
References
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Journal: Endocrinology (2013): 205
Authors: Al-Musawi BM, Al-Allawi N, Abdul-Majeed BA, Eissa AA, Jubrael JM, Hamamy H.
Journal: BMC Blood Disord (2012): 4
Authors: Millimono TS, Loua KM, Rath SL, Relvas L, Bento C, Diakite M, Jarvis M, Daries N, Ribeiro LM, Manco L, Kaeda JS.
Journal: Hemoglobin (2012): 25
Authors: Schneider AM, Rawat D, Weinstein LS, Gupte SA, Richards WO.
Journal: Surg Endosc (2012): 823
Authors: Shah SS, Diakite SA, Traore K, Diakite M, Kwiatkowski DP, Rockett KA, Wellems TE, Fairhurst RM.
Journal: Sci Rep (2012): 299
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