Description
Hexokinase/Glucokinase (HK/GK) Activity Assay Kit
The Assay Genie Hexokinase/Glucokinase (HK/GK) Activity Assay Kit measures total hexokinase activity (EC 2.7.1.1), including glucokinase (hexokinase IV, EC 2.7.1.2), in cell and tissue lysates. Hexokinases phosphorylate glucose to glucose-6-phosphate, the first and committing step of glycolysis. HK2 is highly expressed in many cancers, while glucokinase acts as the glucose sensor of pancreatic β-cells and hepatocytes.
Glucose-6-phosphate formed by HK/GK is coupled to NADPH production, which reduces the tetrazolium dye INT to a coloured formazan read at 492 nm. The assay uses 20 mM glucose, a concentration high enough to capture low-affinity glucokinase together with HK1–3, and calculates activity directly in IU/L.
| Product Name: | Hexokinase/Glucokinase Assay Kit |
| SKU: | BR00009 |
| Kit Size: | 100 wells |
| Detection Method: | Colorimetric (NADPH-coupled INT → formazan, OD 492 nm) |
| Sample Types: | Cell and tissue lysates |
| Recommended Protein: | 0.2–2 mg/ml |
| Incubation Time: | 60 or 90 min at 37 °C |
| Units: | IU/L (µmol/L/min); normalise to protein or GAPDH |
| Equipment Required: | Microplate reader (492 nm) |
| Storage: | -80 °C (see component storage) |
| Intended Use: | For research use only (RUO). Not for diagnostic procedures. |
- Measures total HK activity, including glucokinase (20 mM glucose)
- Low sample volume: 10 µl per well
- Direct activity calculation in IU/L, no standard curve required
- Per-sample no-substrate control
- Simple 96-well mix-and-read format
- HK/GK phosphorylates glucose to glucose-6-phosphate.
- Glucose-6-phosphate is coupled to NADPH production, which drives the reduction of INT to a coloured formazan. Formazan has an absorbance maximum at 492 nm (ε = 18 mM-1cm-1), so activity is calculated directly in IU/L (µmol/L/min) without a standard curve.
- Activity is calculated from ΔOD (reaction − control): IU/L = ΔOD × 13.89 (60 min) or × 9.26 (90 min).
- Cells: ~105 washed cells in 50–100 µl ice-cold 1x Cell Lysis Solution, 5 min on ice.
- Tissue: ~5 mg tissue homogenised in 0.5 ml ice-cold 1x Cell Lysis Solution.
- Centrifuge at ~14,000 rpm for 5 min; adjust protein to 0.2–2 mg/ml.
| Component | Size | Storage |
| HK Assay Solution | 5 ml | -80 °C, protect from light |
| 50x HK Substrate Solution | 0.5 ml | 4 °C |
| 10x Cell Lysis Solution | 25 ml | Room temperature |
- Glycolysis and glucose uptake/utilisation studies
- Cancer metabolism (HK2) and the Warburg effect
- Diabetes, β-cell glucose sensing and hepatic glucokinase research
- Screening of hexokinase inhibitors or glucokinase activators
Does this kit measure glucokinase as well as hexokinase?
Yes. The assay uses 20 mM glucose, so it measures total activity including low-affinity glucokinase (HK IV) as well as HK1–3.
How much sample do I need?
Only 10 µl of lysate per well, at 0.2–2 mg/ml protein.
How is HK activity calculated?
ΔOD at 492 nm (reaction − control) × 13.89 for 60 min or × 9.26 for 90 min, giving IU/L.
