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Amplite® Fluorimetric Glucose-6-Phosphate Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare G6P working solution (50 µL)
- Add G6P standards or test samples (50 µL)
- Incubate at room temperature for 30 minutes - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Important notes
Thaw kit components 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. G6P standard solution (100 mM):
Add 100 µL of H2O or 1x PBS buffer into the vial of G6P Standard (Component D) to make 100 mM G6P standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13804
Add 10 µL of 100 mM G6P standard solution into 990 µL 1x PBS buffer to generate 1 mM G6P standard solution. Then, add 100 µL of 1 mM G6P standard solution into 900 µL 1 x PBS buffer to make 100 µM G6P standard solution (G6P7). Take 100 µM G6P standard solution (G6P7) and perform 1:3 serial dilutions to get serially diluted G6P standards (G6P6 - G6P1) with 1x PBS buffer. Note: Diluted G6P 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 G6P working solution. Note: This G6P working solution is enough for one 96-well plate.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of G6P standards and test samples in a solid black 96-well microplate. G6P=G6P Standards (G6P1 - G6P7, 0.14 to 100 µM), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| G6P1 | G6P1 | ... | ... |
| G6P2 | G6P2 | ... | ... |
| G6P3 | G6P3 | ||
| G6P4 | G6P4 | ||
| G6P5 | G6P5 | ||
| G6P6 | G6P6 | ||
| G6P7 | G6P7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| G6P1 - G6P7 | 50 µL | Serial Dilution (0.14 to 100 µM) |
| BL | 50 µL | Dilution Buffer |
| TS | 50 µL | test sample |
- Prepare G6P standards (G6P), 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 G6P working solution to each well of G6P standard, blank control, and test samples to make the total G6P assay volume of 100 µL/well. For a 384-well plate, add 25 µL of G6P 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 fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, Cutoff = 570nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the ratio of A575nm/A605nm. The absorption detection has lower sensitivity compared to fluorescence reading.
References
Authors: Legeza B, Balazs Z, Nashev LG, Odermatt A.
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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