Description
Glutamate Dehydrogenase Activity Assay Kit (BA0025) (BA0025)
The Glutamate Dehydrogenase Activity Assay Kit (SKU: BA0025) is a non-radioactive, colorimetric assay for the quantitative determination of glutamate dehydrogenase (GLDH) activity in biological samples. GLDH catalyses the interconversion of glutamate and alpha-ketoglutarate, and elevated serum levels are an important indicator of liver damage, particularly when interpreted alongside the aminotransferases. In this kit GLDH activity is coupled through an NADH-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 0.4 to 80 U/L over a thirty-minute reaction and a detection limit of 0.1 U/L for longer incubations. The homogeneous format is convenient and readily automated for high-throughput work across plasma, serum, urine, tissue and culture media.
| Product Name: | Glutamate Dehydrogenase Activity Assay Kit (BA0025) |
| SKU: | BA0025 |
| Detection Method: | Colorimetric kinetic; NADH-coupled reduction of MTT (OD565nm) |
| Detection Range: | 0.4 to 80 U/L for a 30-minute reaction; detection limit 0.1 U/L for 120-minute reaction (20 µL sample) |
| Sample Type: | Plasma, serum, urine, tissue, culture media |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 12 months after receipt. |
| Shipping: | Room Temperature |
A non-radioactive, colorimetric kinetic assay for the quantitative determination of glutamate dehydrogenase (GLDH) activity. GLDH activity is coupled through an NADH-linked reaction to the reduction of MTT; the increase in absorbance at 565 nm is directly proportional to enzyme activity. Linear detection range is 0.4 to 80 U/L for a 30-minute reaction.
- Fast and sensitive: linear detection range 0.4 to 80 U/L for a 30-minute reaction, with a detection limit of 0.1 U/L for a 120-minute reaction.
- Convenient and high-throughput: homogeneous mix-incubate-measure format, readily automated on HTS liquid-handling systems.
- GLDH activity determination in biological samples such as plasma, serum, urine, 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 (WR) per 96-well assay by mixing 10 µL Substrate, 14 µL MTT Solution, 9 µL NAD Solution, 1 µL Diaphorase and 50 µL Assay Buffer. Prepare Blank Working Reagent (BWR) identically but without Substrate (60 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 sample into two separate wells; add 80 µL WR to one and 80 µL BWR to the other. Tap briefly to mix. |
| 5 | Read OD565nm (OD0) immediately and again after 30 min (OD30). |
Subtract OD0 from OD30 for each sample and sample blank to obtain ΔODs and ΔODb. GLDH Activity = (ΔODs - ΔODb) / t x [273 / (ODCAL - ODH2O)] x n (U/L), where t is the reaction time (30 min recommended) and n is the dilution factor. One unit converts 1 µmole of glutamate to alpha-ketoglutarate per minute at pH 8.2.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Diaphorase | 120 µL | -20°C |
| NAD Solution | 1 mL | -20°C |
| Calibrator | 1.5 mL | -20°C |
| MTT Solution | 1.5 mL | -20°C |
| Substrate (1 M Glutamate) | 1.5 mL | -20°C |
