Description
Isocitrate Dehydrogenase Assay Kit (BA0038) (BA0038)
The Isocitrate Dehydrogenase Assay Kit (SKU: BA0038) is a non-radioactive, colorimetric assay for the quantitative determination of isocitrate dehydrogenase (IDH) activity in biological samples. IDH catalyses the interconversion of isocitrate and alpha-ketoglutarate; there are three isoforms, and this kit measures the activity of the NADP-dependent IDH1 and IDH2 isoforms, whose mutations have been linked with various brain tumours and acute myeloid leukaemia. IDH 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 directly proportional to enzyme activity, giving a linear range of 0.1 to 100 U/L over a thirty-minute reaction at 37°C. The homogeneous format is convenient and readily automated for high-throughput work across plasma, serum, tissue and culture media.
| Product Name: | Isocitrate Dehydrogenase Assay Kit (BA0038) |
| SKU: | BA0038 |
| Detection Method: | Colorimetric kinetic; NADPH-coupled reduction of MTT (OD565nm) |
| Detection Range: | 0.1 to 100 U/L for a 30-minute reaction (20 µL sample) |
| Sample Type: | Plasma, serum, tissue, culture media |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at 37°C |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 13 months after receipt. |
| Shipping: | Room Temperature |
A non-radioactive, colorimetric kinetic assay for the quantitative determination of isocitrate dehydrogenase (IDH) activity, measuring the NADP-dependent isoforms. IDH activity is coupled through an NADPH-linked reaction to the reduction of MTT; the increase in absorbance at 565 nm is directly proportional to enzyme activity. Linear detection range is 0.1 to 100 U/L for a 30-minute reaction.
- Fast and sensitive: linear detection range 0.1 to 100 U/L for a 30-minute reaction (20 µL sample).
- Convenient and high-throughput: homogeneous mix-incubate-measure format, readily automated on HTS liquid-handling systems.
- IDH activity determination in biological samples such as plasma, serum, 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: keep thawed diaphorase on ice and equilibrate other reagents to 37°C. Prepare Working Reagent per well by mixing 9 µL Substrate, 9 µL NADP/MTT Solution, 1 µL Diaphorase and 70 µL Assay Buffer; fresh reconstitution is recommended. |
| 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 | Incubate at 37°C and read OD565nm at 10 minutes (OD10) and again at 30 minutes (OD30). |
Subtract OD10 from OD30 for each sample to compute ΔODs. IDH Activity = (ΔODs / t) x [273 / (ODCAL - ODH2O)] x n (U/L), where t is the reaction time (20 min) and n is the dilution factor. One unit converts 1 µmole of isocitrate to alpha-ketoglutarate per minute at 37°C and 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 |
