Description
Hexokinase Assay Kit (BA0248) (BA0248)
The Hexokinase Assay Kit (SKU: BA0248) is a non-radioactive, colorimetric assay for the quantitative kinetic determination of hexokinase activity in biological samples. Hexokinase (EC 2.7.1.1) is a mitochondrial and cytosolic enzyme catalysing the first committed step of glycolysis, in which hexose sugars, mainly glucose, are phosphorylated using ATP to form glucose-6-phosphate, and is implicated in numerous human diseases including cancer and diabetes. The assay involves a stepwise reaction based on the reduction of the tetrazolium salt MTT in an NADPH-coupled enzymatic reaction to a coloured product with an absorption maximum at 565 nm. The increase in absorbance at 565 nm is proportional to the enzyme activity, with a linear detection range of 0.22 to 100 U/L for a 15-minute reaction using a 20 µL sample. This homogeneous mix-incubate-measure assay is readily automated on high-throughput liquid-handling systems for thousands of samples per day.
| Product Name: | Hexokinase Assay Kit (BA0248) |
| SKU: | BA0248 |
| Detection Method: | Colorimetric kinetic (OD565nm) |
| Detection Range: | 0.22 to 100 U/L |
| Sample Type: | Microorganisms, plasma, serum, cultured cells, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 15 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A quantitative colorimetric kinetic assay for hexokinase activity. Hexokinase activity is measured through an NADPH-coupled reduction of the tetrazolium salt MTT to a coloured product with an absorption maximum at 565 nm.
- Fast and sensitive. Linear detection range (20 µL sample): 0.22 to 100 U/L for a 15 min reaction.
- Convenient. Homogeneous mix-incubate-measure type assay.
- High-throughput. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- Hexokinase activity determination in biological samples (e.g. microorganisms, plasma, serum, cultured cells, tissue and culture media)
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent should be quick and mixing brief but thorough; a multi-channel pipettor is recommended. Assays may be run at any desired temperature (e.g. 25°C or 37°C). |
| 2 | Sample preparation: serum and plasma are assayed directly. Tissue: rinse in phosphate buffered saline (pH 7.4) to remove blood, homogenise 50 mg tissue in ~200 µL buffer containing 50 mM potassium phosphate (pH 7.5), centrifuge at 10,000 × g for 15 min at 4°C and remove supernatant. Cell lysate: collect cells by centrifugation at 2,000 × g for 5 min at 4°C (use a cell scraper for adherent cells), homogenise or sonicate in cold buffer containing 50 mM potassium phosphate (pH 7.5), centrifuge at 10,000 × g for 15 min at 4°C and remove supernatant. Samples may be stored at -20 to -80°C for at least one month. |
| 3 | Reagent preparation: equilibrate reagents to the desired reaction temperature and briefly centrifuge tubes. Prepare enough Working Reagent (WR) for all wells by mixing, for each 96-well assay: 8 µL Substrate Mix, 8 µL NADP/MTT solution, 1 µL Enzyme A, 1 µL Enzyme B and 70 µL Assay Buffer. |
| 4 | Transfer 100 µL H2O (OD_H2O) and 100 µL Calibrator (OD_CAL) into wells of a clear flat-bottom 96-well plate. |
| 5 | Transfer 20 µL of each sample into separate wells, then add 80 µL WR to each sample well. Tap the plate briefly to mix. |
| 6 | Read OD565nm (OD0), and again after 15 min (OD15) on a plate reader. |
Subtract OD0 from OD15 for each sample to compute ∆ODS. Hexokinase activity = (∆ODS / (ODCAL – ODH2O)) × (Reaction Vol / (t × Sample Vol)) × 273 × n (U/L), where 273 relates to the molar absorption coefficient of reduced MTT and light pathlength calculated from the calibrator, ODCAL and ODH2O are the OD565nm (OD0) values of the calibrator and water, t is the reaction time (15 min recommended), Reaction Vol and Sample Vol are 100 µL and 20 µL respectively, and n is the dilution factor. Unit definition: 1 U of hexokinase catalyses the conversion of 1 µmole of glucose to glucose-6-phosphate per min at pH 8.2. If activity exceeds 100 U/L, use a shorter reaction time or dilute the sample; for activity < 1 U/L, extend incubation to 2 hours.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| NADP/MTT | 1 mL | -20°C |
| Substrate Mix | 1 mL | -20°C |
| Enzyme A | 120 µL | -20°C |
| Enzyme B | 120 µL | -20°C |
| Calibrator | 1 mL | -20°C |
