Amplite® Colorimetric Fructose-6-Phosphate Activity Assay Kit

The Amplite® Colorimetric Fructose-6-Phosphate Assay Kit is a highly sensitive and simple method of quantifying F6P present in a variety of samples. This kit uses a coupled enzyme assay where F6P, in the presence of ATP, is converted to fructose-1,6-diphosphate and ADP by the PFK enzyme. The aldolase enzyme then converts fructose-1,6-diphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Subsequently, glyceraldehyde-3-phosphate dehydrogenase, in the presence of NAD, converts glyceraldehyde-3-phosphate into glyceraldehyde-1,3-bisphosphate and NADH. The NADH produced then reduces a colorless substrate to a colored product (A450 nm), which is proportional to the fructose-6-phosphate present. One unit (U) is the amount of enzyme that catalyzes the reaction of 1 µmol of substrate per minute. Fructose-6-phosphate (F6P) is an important intermediate generated during glycolysis. It is produced by the isomerization of glucose-6-phosphate by phosphoglucose isomerase. It serves as a precursor to fructose 1,6-bisphosphate. Beyond its role in energy production, F6P can also be shunted to the non-oxidative pentose phosphate pathway as a means of generating pentose phosphates for nucleotide production. F6P levels surge in rapidly proliferating cells like cancer cells, making it a potential metabolic biomarker in diseases.

Example protocol

AT A GLANCE

Important Note

Thaw all the kit components at room temperature before starting the experiment.

Protocol Summary

Prepare the test samples, and the serially diluted F69 standards (50 μL).
Add the F6P working solution (50 μL).
Incubate for 10-30 minutes at room temperature.
Measure the absorbance at 450 nm.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Fructose-6-Phosphate (F6P) Standard

Add 330 µL of PBS buffer to the vial containing the Fructose-6-Phosphate (F6P) standard (Component F) to prepare a 20 mM (50 nmol/µL) F6P stock solution.

Note: Store the solution at -20°C. Avoid repeated freeze/thaw cycles.

ATP Stock Solution

Add 100 µL of ddH2O to the vial of ATP (Component E) to create a 100X ATP stock solution.

Note: Store this solution at -20°C and avoid repeated freeze/thaw cycles.

F6P Developer Stock Solution

Add 100 µL of ddH2O to the vial of F6P Developer (Component D) to create a 100X F6P Developer stock solution.

Note: Store this solution at -20°C and avoid repeated freeze/thaw cycles.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11330

F6P Standard

Add 5 μL of the 20 mM F6P standard solution to 495 μL of PBS Buffer to create a 200 μM F6P solution, STD7. Next, take 150 μL of the STD7 solution and perform a 1:2 serial dilution in PBS Buffer to generate a series of diluted F6P standards (STD7 to STD1).

PREPARATION OF WORKING SOLUTION

F6P Working Solution

Add 1.0 mL of F6P Probe (Component A) to 4 mL F6P Assay Buffer (Component B), and mix well.
Transfer the 5 mL buffer mixture from above to the F6P Enzyme Mix bottle, and mix well.
Add 50 µL of the ATP stock solution to the same bottle, and mix well.
Add 50 µL of the F6P Developer stock solution to the same bottle, and mix well.

Note: Prepare the F6P working solution fresh before each experiment and protect it from light. A 5 mL preparation is sufficient for 100 tests. Adjust the volume proportionally based on the number of tests you plan to conduct.

Note: Alternatively, one can prepare a 50X stock solution of F6P Enzyme Mix by adding 100 μL of ddH2O to the bottle of F6P Enzyme Mix (Component C) and mix thoroughly to dissolve the enzyme completely. Next, to prepare the F6P working solution, combine the 50X stock solution with the other components listed in the 'F6P Working Solution' section above, following the specified proportions.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of F6P standards and test samples in a 96-well solid black microplate. (STD = F6P Standards (STD1-STD7, 3.125-200 µM), BL = Blank Control, TS = Test Samples)

BL	BL	TS	TS
STD 1	STD 1	...	...
STD 2	STD 2	...	...
STD 3	STD 3
STD 4	STD 4
STD 5	STD 5
STD 6	STD 6
STD 7	STD 7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
STD 1- STD 7	50 µL	Serial Dilutions (3.125 - 200 µM)
BL	50 µL	PBS
TS	50 µL	Test Sample

Prepare the F6P standards (STD1-7), blank controls (BL), and test samples (TS) as outlined in Tables 1 and 2. When using a 384-well plate, add 25 µL of reagent to each well instead of the standard 50 µL.
Add 50 µL of F6P Working Solution to each well containing the F6P standard, blank control, and test samples. If you are using a 384-well plate, add 25 µL of F6P Working Solution to each well instead.
Incubate at room temperature for 10-30 minutes, protected from light.
Monitor the absorbance intensity with an absorbance microplate reader at 450 nm.

References

View all 50 references: Citation Explorer

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.
Authors: Drury, James and Geisen, Mariah E and Tessmann, Josiane Weber and Rychahou, Piotr G and Kelson, Courtney O and He, Daheng and Wang, Chi and Evers, B Mark and Zaytseva, Yekaterina Y
Journal: International journal of molecular sciences (2024)

Silencing of Glutamine: Fructose-6-Phosphate Aminotransferase Impairs Growth and Development in Sogatella furcifera (Hemiptera: Delphacidae).
Authors: Wang, Zhao and Long, Guiyun and Zhu, Huan and Jin, Daochao and Yang, Hong and Zhou, Cao
Journal: Biomolecules (2023)

Dry-milled flour rice 'Seolgaeng' harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2.
Authors: Kang, Ju-Won and Lee, Sang-Kyu and Shim, Su-Hyeon and Shin, Dongjin and Cho, Jun-Hyeon and Lee, Ji-Yoon and Ko, Jong-Min and Ji, Hyeonso and Park, Hyang-Mi and Ahn, Eok-Keun and Lee, Jong-Hee and Jeon, Jong-Seong
Journal: Frontiers in plant science (2023): 1231914

Cancer-associated fibroblast expression of glutamine fructose-6-phosphate aminotransferase 2 (GFPT2) is a prognostic marker in gastric cancer.
Authors: Yang, Shuo and Li, Guoli and Yin, Xin and Wang, Yufei and Jiang, Xinju and Bian, Xiulan and Fang, Tianyi and Yin, Shengjie and Zhang, Lei and Xue, Yingwei
Journal: The journal of pathology. Clinical research (2023): 391-408

Fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase regulates energy metabolism and synthesis of storage products in developing rice endosperm.
Authors: Chen, Xiaoli and Ji, Yi and Zhao, Weiying and Niu, Huanying and Yang, Xue and Jiang, Xiaokang and Zhang, Yipeng and Lei, Jie and Yang, Hang and Chen, Rongbo and Gu, Chuanwei and Xu, Hongyi and Dong, Hui and Duan, Erchao and Teng, Xuan and Wang, Yunlong and Zhang, Yuanyan and Zhang, Wenwei and Wang, Yihua and Wan, Jianmin
Journal: Plant science : an international journal of experimental plant biology (2023): 111503

Page updated on May 19, 2025

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H-phrase	H303, H313, H333
Hazard symbol	XN
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Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200