Description
Glutathione Reductase Assay Kit (BA0087) (BA0087)
The Glutathione Reductase Assay Kit (SKU: BA0087) provides a non-radioactive, colorimetric procedure for accurately measuring glutathione reductase activity in biological samples. Glutathione reductase reduces oxidised glutathione to the reduced sulfhydryl form, which is an important cellular antioxidant, and a high ratio of reduced to oxidised glutathione is important for protection against oxidative stress. This assay uses Ellman's method, in which DTNB reacts with the reduced glutathione generated from the reduction of oxidised glutathione to form a yellow product. The rate of change in optical density, measured at 412 nm, is directly proportional to the glutathione reductase activity in the sample.
| Product Name: | Glutathione Reductase Assay Kit (BA0087) |
| SKU: | BA0087 |
| Detection Method: | Colorimetric (kinetic) |
| Detection Range: | 0.4 to 50 U/L |
| Sample Type: | Plasma, serum, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 20 to 30 minutes at the desired temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric kinetic determination of glutathione reductase activity. DTNB reacts with reduced glutathione generated by the enzyme to form a yellow product; the rate of optical density change at 412 nm is proportional to activity.
- Fast and sensitive; linear detection range 0.4 to 50 U/L with a 20 uL sample, detection limit 0.4 U/L
- Convenient and high-throughput mix-incubate-measure format
- Readily automated on liquid handling systems
- Assays can be run at any desired temperature, such as 25C or 37C
- Glutathione reductase 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 | This assay is a kinetic reaction; add working reagent quickly and mix briefly but thoroughly, using a multi-channel pipettor. Assays can be run at any desired temperature (e.g. 25C or 37C). |
| 2 | Sample preparation. Assay serum and plasma directly. For tissue, rinse in phosphate-buffered saline, homogenise 50 mg in about 200 uL 50 mM potassium phosphate (pH 7.5), centrifuge at 10,000 x g for 15 minutes at 4C and use the supernatant. For cell lysate, collect cells by centrifugation, homogenise or sonicate in cold 50 mM potassium phosphate (pH 7.5) and centrifuge as above. Samples can be stored at -20 to -80C for at least one month. |
| 3 | Calibrator. Transfer 100 uL Calibrator and 100 uL Assay Buffer to separate wells in a 96-well plate. |
| 4 | Reagent preparation. Equilibrate reagents to the desired reaction temperature and briefly centrifuge tubes. Prepare working reagent by mixing, per 96-well assay, 8 uL Substrate, 8 uL Cosubstrate, 1 uL GDH, 0.5 uL DTNB and 70 uL Assay Buffer. |
| 5 | Transfer 20 uL of each sample into separate wells and add 80 uL working reagent to each sample well; tap briefly to mix. |
| 6 | Incubate the plate at the desired temperature and read OD412nm at 10 minutes (OD10) and again at 30 minutes (OD30) on a plate reader. |
Subtract OD10 from OD30 for each sample to obtain dODs. GR activity (U/L) = (dODs / t) x (440 / (OD-CAL - OD-buffer)) x n, where OD-CAL and OD-buffer are the OD412nm values of the Calibrator and Assay Buffer, t is the reaction time (20 min recommended) and n is the dilution factor. One unit of GR catalyses the conversion of 1 umole of oxidised glutathione to 2 umole reduced glutathione per minute at pH 7.6. If activity exceeds 50 U/L, use a shorter reaction time or dilute the sample and repeat.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| Substrate | 1 mL | -20C |
| Cosubstrate | 1 mL | -20C |
| GDH | 120 uL | -20C |
| DTNB | 60 uL | -20C |
| Calibrator | 1.5 mL | -20C |
