Amplite® Colorimetric Glucose-6-Phosphate Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Platform
Absorbance microplate reader
Absorbance | 575/605 nm |
Recommended plate | Clear bottom |
Components
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 absorbance ratio increase at A575nm/A605nm
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 stock 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/13805
Add 10 µL of 100mM 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 1x PBS buffer to make 100 µM G6P standard solution (G6P7). Take 100 µM G6P standard solution (G6P7) and perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted G6P standards (G6P6 - G6P1). 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. 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 G6P standards and test samples in a white clear bottom 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 Dilutions (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 absorbance ratio increase with an absorbance plate reader at A575nm/A605nm.
Images
Citations
Authors: Xu, Li-Hui and Duan, Huan-ren and Zhang, Yu and Zhang, Tong-jia and Liu, Ya-shu and Zhou, Jin-sa and Hu, Wen-xiu and Zhang, Huan and Shi, Wan-ying and Sun, Su-ju
Journal: (2022)
Authors: Reyes, Carolina and Poulin, Alexandre and Nystr{\"o}m, Gustav and Schwarze, Francis WMR and Ribera, Javier
Journal: Journal of Fungi (2021): 222
References
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Journal: Sci Rep (2012): 299
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Journal: Am J Physiol Endocrinol Metab (2012): E959
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Authors: Nadarajan V, Shanmugam H, Sthaneshwar P, Jayaranee S, Sultan KS, Ang C, Arumugam S.
Journal: Int J Lab Hematol (2011): 463
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