Amplite® Colorimetric Hexokinase Assay Kit

Amplite® Colorimetric Hexokinase Assay Kit is a colorimetric assay kit for measuring hexokinase activity through a coupled NADH-dependent readout in a microplate format.

Colorimetric detection: Measures a colored product generated through an NADH-dependent coupled enzyme reaction.
Rapid workflow: Detects hexokinase activity after a 10-30 minute incubation.
Coupled enzyme chemistry: Links glucose phosphorylation to glucose-6-phosphate oxidation and NADH production.
Microplate compatible: Read absorbance at 450 nm using a clear-bottom microplate.
Applications: Suitable for hexokinase activity measurements in glycolysis and cellular metabolism research.

The NADPH dose response was measured using the Amplite® Colorimetric Hexokinase Assay Kit on a 96-well clear-bottom microplate. After a 10-minute incubation, readings were taken at 450 nm with a ClarioStar microplate reader (BMG).

Protocol

SDS

COA

Catalog	Size	Price	Quantity
11319	100 Tests	Price

Overview

The Amplite® Colorimetric Hexokinase Assay Kit is designed to accurately measure the activity of hexokinases from various sources using a simple and quick protocol. This kit utilizes a coupled enzyme assay, where glucose is phosphorylated to glucose-6-phosphate and subsequently oxidized to produce NADH. The resulting NADH reduces a colorless substrate to a colored product, with absorbance measured at 450 nm, which is proportional to the hexokinase present in the samples. One unit (U) is the amount of enzyme that catalyzes the reaction of 1 µmol of substrate per minute. Hexokinases are intracellular enzymes that mediate ATP-dependent phosphorylation of hexoses (e.g., glucose, mannose, and fructose) to their respective hexose 6-phosphates. These phosphate esters can either be broken down to pyruvate by glycolysis or used for different biochemical/biosynthesis pathways. There are four isozymes of hexokinase known in vertebrates, Hexokinase I (A), II (B), III (C), and IV (D), which have different intracellular and tissue distribution, and kinetic characteristics. Changes in hexokinase level and activity are associated with diseases like X-linked muscular dystrophy, chronic non-spherocytic hemolytic anemia, insulin resistance, and cancer.

References

View all 49 references: Citation Explorer

The Role of Hexokinases in Epigenetic Regulation: Altered Hexokinase Expression and Chromatin Stability in Yeast.

Authors:

Karri, Srinivasu and Dickinson, Quinn and Jia, Jing and Gan, Haiyun and Wang, Zhiquan and Deng, Yibin and Yu, Chuanhe

Journal:

Research square (2024)

A gain of function mutation in AKT1 increases hexokinase 2 and diminishes oxidative stress in meningioma.

Authors:

Singh, Swati and Lathoria, Kirti and Umdor, Sonia B and Singh, Jyotsna and Suri, Vaishali and Sen, Ellora

Journal:

Cytokine (2024): 156535

Glioma hexokinase 3 positively correlates with malignancy and macrophage infiltration.

Authors:

Liang, Tingyu and Zhou, Xingang and Wang, Yu and Ma, Wenbin

Journal:

Metabolic brain disease (2024)

LC/MS/MS and GC/MS/MS metabolic profiling of Leontodon hispidulus, in vitro and in silico anticancer activity evaluation targeting hexokinase 2 enzyme.

Authors:

Abd-El-Aziz, Noha Mokhtar and Hifnawy, Mohamed Saeed and Lotfy, Rehab Ahmed and Younis, Inas Youssef

Journal:

Scientific reports (2024): 6872

Abatement of the binding of human hexokinase II enzyme monomers by in-silico method with the design of inhibitory peptides.

Authors:

Karamifard, Faranak and Mazaheri, Mahta and Dadbinpour, Ali

Journal:

In silico pharmacology (2024): 30

Usage and storage

Intended use

Research Use Only (RUO)

Instrument settings

Absorbance microplate reader
Absorbance	450 nm
Recommended plate	Clear bottom

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Page updated on October 24, 2026

Component A: HK Assay Buffer	4 mL
Component B: HK Probe	1 mL
Component C: HK Enzyme Mix	1 Bottle
Component D: HK Substrate	1 Vial
Component E: HK Coenzyme	1 Vial
Component F: Hexokinase Positive Control (5 µg)	1 Vial
Component G: NADPH Standard (335 µg)	1 Vial