Description
Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit (BA0027) (BA0027)
The Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit (SKU: BA0027) is a non-radioactive, colorimetric assay for the quantitative determination of G6PDH activity in biological samples. G6PDH is a cytosolic enzyme of the pentose phosphate pathway that supplies reducing power to cells by maintaining NADPH levels, reducing NADP to NADPH while oxidising glucose-6-phosphate. A genetic deficiency of G6PDH predisposes to non-immune haemolytic anaemia, making its measurement of considerable research interest. In this kit G6PDH activity is coupled through an NADPH-linked reaction to the reduction of the tetrazolium salt MTT, producing a reduced form with an absorption maximum at 565 nm. The increase in absorbance at 565 nm is proportional to enzyme activity, with a linear range of 0.2 to 100 U/L over a fifteen-minute reaction. The homogeneous format is convenient and readily automated for high-throughput work.
| Product Name: | Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit (BA0027) |
| SKU: | BA0027 |
| Detection Method: | Colorimetric kinetic; NADPH-coupled reduction of MTT (OD565nm) |
| Detection Range: | 0.2 to 100 U/L for a 15-minute reaction (20 µL sample) |
| Sample Type: | Plasma, serum, erythrocytes, tissue, culture media |
| Species Reactivity: | All |
| Assay Time: | 15 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 15 months after receipt. |
| Shipping: | Room Temperature |
A non-radioactive, colorimetric kinetic assay for the quantitative determination of glucose-6-phosphate dehydrogenase (G6PDH) activity. G6PDH activity is coupled through an NADPH-linked reaction to the reduction of MTT; the increase in absorbance at 565 nm is proportional to enzyme activity. Linear detection range is 0.2 to 100 U/L for a 15-minute reaction.
- Fast and sensitive: linear detection range 0.2 to 100 U/L for a 15-minute reaction (20 µL sample).
- Convenient and high-throughput: homogeneous mix-incubate-measure format, readily automated on HTS liquid-handling systems.
- G6PDH activity determination in biological samples such as plasma, serum, erythrocytes, 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 | Sample preparation: assay serum and plasma directly. Homogenise tissue (50 mg) in ~200 µL 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 g for 15 min at 4°C; prepare cell lysates similarly. Samples may be stored at -20 to -80°C for at least one month. |
| 2 | Reagent preparation: prepare Working Reagent per 96-well assay by mixing 8 µL Substrate, 8 µL NADP/MTT Solution, 1 µL Diaphorase and 70 µL Assay Buffer. |
| 3 | Transfer 100 µL H2O and 100 µL Calibrator into wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 20 µL of each sample into separate wells and add 80 µL Working Reagent to each. Tap briefly to mix. |
| 5 | Read OD565nm (OD0) immediately and again after 15 min (OD15). |
Subtract OD0 from OD15 for each sample to compute ΔODs. G6PDH Activity = (ΔODs / t) x [273 / (ODCAL - ODH2O)] x n (U/L), where t is the reaction time (15 min recommended) and n is the dilution factor. One unit converts 1 µmole of NADP to NADPH per minute at pH 8.2.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Diaphorase | 120 µL | -20°C |
| NADP/MTT | 1 mL | -20°C |
| Calibrator | 1.5 mL | -20°C |
| Substrate | 1 mL | -20°C |
