Description
Catalase Assay Kit (Colormetric/Fluorometric) (BA0083) (BA0083)
The Catalase Assay Kit (Colorimetric/Fluorometric) (SKU: BA0083) provides a simple, direct and high-throughput procedure for measuring catalase activity. Catalase is a ubiquitous antioxidant enzyme that catalyses the decomposition of hydrogen peroxide to water and oxygen, protecting cells from excessive hydrogen peroxide build-up; deficiency has been associated with grey hair and the peroxisomal disorder acatalasia. This improved assay directly measures catalase degradation of hydrogen peroxide using a redox dye. The change in colour intensity at 570 nm, or fluorescence intensity at excitation/emission 530/585 nm, is directly proportional to the catalase activity in the sample.
| Product Name: | Catalase Assay Kit (Colormetric/Fluorometric) (BA0083) |
| SKU: | BA0083 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 0.2 to 5 U/L; fluorometric 0.02 to 0.5 U/L |
| Sample Type: | Serum, plasma, urine, saliva, cell culture, tissue and cells |
| Species Reactivity: | All |
| Assay Time: | 30 minute reaction followed by 10 minute detection |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of catalase activity. Catalase degrades hydrogen peroxide, and the remaining hydrogen peroxide is measured with a redox dye read at 570 nm or by fluorescence at excitation/emission 530/585 nm.
- Sensitive and accurate; uses 10 uL sample
- Colorimetric linear detection range 0.2 to 5 U/L
- Fluorometric linear detection range 0.02 to 0.5 U/L
- Simple and convenient add-incubate-read procedure
- Direct assays of catalase activity in biological samples such as serum, plasma, urine, saliva and cell culture
- Drug discovery and pharmacology: effects of drugs on catalase activity
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. Homogenise tissue (10 mg) and cells (10^6) in 200 uL cold PBS and centrifuge for 10 minutes at 14,000 rpm; use the clear supernatant. Keep SH-containing reagents below 10 uM in the sample. |
| 2 | Reagent preparation. Equilibrate all components to room temperature and briefly centrifuge tubes; keep thawed HRP Enzyme on ice. Use a clear flat-bottom plate for colorimetric assays and a solid black plate for fluorometric assays. Transfer 10 uL sample into wells and prepare a sample blank well with 10 uL Assay Buffer. Add 400 uL Assay Buffer to the Positive Control tube and transfer 10 uL into separate wells. |
| 3 | Enzyme reaction. Mix 5 uL 3% H2O2 and 914 uL distilled water (final 4.8 mM). Prepare 50 uM H2O2 substrate by mixing, per well, 1 uL of 4.8 mM H2O2 with 95 uL Assay Buffer (prepare fresh). Add 90 uL of the 50 uM substrate to the sample, positive control and sample blank wells to initiate the reaction, tap to mix and incubate 30 minutes at room temperature. |
| 4 | H2O2 standard curve. Mix 40 uL of the 4.8 mM H2O2 with 440 uL distilled water to yield 400 uM H2O2 and prepare standards as shown in the dilution table. Transfer 10 uL standards into separate wells and add 90 uL Assay Buffer. |
| 5 | Detection. Prepare Detection Reagent by mixing, per reaction well, 102 uL Assay Buffer, 1 uL Dye Reagent and 1 uL HRP Enzyme. At the end of the 30 minute incubation, add 100 uL Detection Reagent per well, tap to mix and incubate for 10 minutes. |
| 6 | Read optical density at 570 nm (550-585 nm). For the more sensitive fluorescence assay (585/530 nm), use 20, 12, 6 and 0 uM H2O2 for the standard curve. |
Subtract the blank value (#4) from the standard values and plot dOD or dF against standard concentrations to obtain the slope. Catalase (U/L) = [(R-sample blank - R-sample) / (slope x 30 min)] x n, where R-sample blank and R-sample are the optical density or fluorescence readings of the sample blank and sample, 30 min is the reaction time and n is the dilution factor. One unit decomposes 1 umole of hydrogen peroxide per minute at pH 7.0 and room temperature.
| Component | Quantity | Storage |
| Assay Buffer | 25 mL | -20C |
| HRP Enzyme | 120 uL | -20C |
| Dye Reagent | 120 uL | -20C |
| H2O2 Solution (3%) | 100 uL | -20C |
| Positive Control (Catalase) | 8 uL | -20C |
