Description
Superoxide Dismutase Activity Assay Kit (BA0150) (BA0150)
The Superoxide Dismutase Activity Assay Kit (SKU: BA0150) provides a convenient colorimetric means for the quantitative determination of superoxide dismutase (SOD) activity in biological samples. Superoxide dismutases catalyse the dismutation of superoxide into oxygen and hydrogen peroxide and form an important antioxidant defence in all cells exposed to oxygen, with aberrant activity linked to amyotrophic lateral sclerosis, neural disorders and cancer. In this assay superoxide is produced by a xanthine oxidase catalysed reaction and reacts with a WST-1 dye to form a coloured product; SOD scavenges the superoxide, so less is available for the chromogenic reaction. The colour intensity at 440 nm is used to determine the SOD activity in the sample. The homogeneous mix-incubate-measure format is readily automated for high-throughput use.
| Product Name: | Superoxide Dismutase Activity Assay Kit (BA0150) |
| SKU: | BA0150 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.05 - 3 U/mL SOD |
| Sample Type: | Blood, cell, tissue and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at -20 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A colorimetric assay for the quantitative determination of superoxide dismutase activity. Xanthine oxidase generates superoxide, which reacts with a WST-1 dye to form a coloured product measured at 440 nm; SOD reduces this signal by scavenging superoxide. The homogeneous format requires no wash or transfer steps and is suitable for high-throughput screening.
- Sensitive and accurate, with a linear detection range of 0.05 - 3 U/mL SOD
- Convenient homogeneous mix-incubate-measure format
- No wash or transfer steps required
- Readily automated on HTS liquid-handling systems
- Determination of SOD in blood, cell, tissue and other biological samples
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 | Prepare samples: homogenise tissue in cold lysis buffer (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X-100) and centrifuge at 12,000 g for 5 min at 4 C; prepare cell lysates similarly. Dilute serum or plasma 1:5 and red cell lysate 1:100 prior to assay. |
| 2 | Bring all reagents to room temperature (25 C); briefly vortex the Xanthine tube and keep enzyme tubes on ice. |
| 3 | Prepare standards: mix 8 uL SOD Enzyme with 392 uL Diluent (3 U/mL) and dilute to 3.0, 2.4, 1.8, 1.2, 0.54, 0.24, 0.12 and 0 U/mL. Transfer 20 uL of each standard and 20 uL of each sample into wells of a clear flat-bottom 96-well plate. |
| 4 | Prepare Working Reagent per well by mixing 160 uL Assay Buffer, 5 uL Xanthine and 5 uL WST-1. Transfer 160 uL to each well and tap to mix. |
| 5 | Prepare diluted XO Enzyme (4 uL XO with 20 uL Diluent per well) and quickly add 20 uL to each well. Tap to mix. |
| 6 | Immediately read optical density at 440 nm (OD0), incubate for 60 min at room temperature in the dark and read again (OD60). |
For each well calculate dOD60 = OD60 - OD0, then ddOD = dOD(Standard 8) - dOD, where Standard 8 has no SOD activity. Plot the standard curve of ddOD against SOD concentration (U/mL) and use the sample ddOD to determine SOD activity from the standard curve.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20 C |
| Diluent | 20 mL | -20 C |
| SOD Enzyme | 120 uL | -20 C |
| XO Enzyme | 400 uL | -20 C |
| Xanthine | 600 uL | -20 C |
| WST-1 | 600 uL | -20 C |
