Catalase (EC 1.11.1.6), is an ubiquitous antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) to water and oxygen
catalase 2 H2O2 O2+2 H2OBy preventing excessive H2O2 build up, catalase allows important cellular processes which produce H2O2 as a byproduct to occur safely. Deficiency in catalase activity has been associated with grey hair and peroxisomal disorder acatalasia. Simple, direct and high-throughput assays for catalase activity find wide Applications:. Improved assay directly measures catalase degradation of H2O2 using a redox dye. The change in color intensity at 570nm or fluorescence intensity (lem/ex=585/530nm) is directly proportional to the catalase activity in the sample.
Key Features:Sensitive and accurate. Use 10ul sample. Linear detection range 0.2 to 5 U/L catalase activity
Simple and Convenient. The procedure involves adding a Substrate to sample, incubation for 30 min, followed by a Detection Reagent and reading the optical density or fluorescence intensity.
Applications:Direct Assays: catalase activity in biological samples e.g. serum, plasma, urine, saliva, cell culture etc
Drug Discovery/Pharmacology: effects of drugs on catalase activity.
Kit ComponentsC2090-09A: Assay Buffer, 1x25mlC2090-09B: HRP Enzyme, 1x120ulC2090-09C: Dye Reagent, 1x120ulC2090-09D: H2O2 Solution, 1x100ul 3% H2O2C2090-09E: Positive Control, 1x8ul CatalaseStorage conditions. The kit is shipped on ice. Store all components at -20°C. Shelf life of 6 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Sample Preparation:Tissue (10 mg) and cells (106) are homogenized in 200ul cold Assay Buffer. Centrifuge 10 min at 14,000 rpm to pellet any debris. Use clear supernatant for assay
Note: SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol) are known to interfere in this assay and should be kept below 10uM in the sample.
Assay Procedure:1. Reagent Preparation. Equilibrate all components to room temperature. Briefly centrifuge all tubes before opening. Keep thawed HRP Enzyme on ice. For Colorimetric Assay:s, use a clear flat-bottom 96-well plate. For Fluorometric Assay:s, use a solid black flat-bottom 96-well plate. Samples and Controls: transfer 10ul sample into wells of the 96-well plate. In addition, for each assay run, prepare one sample blank well that contains only 10ul Assay Buffer. Add 400ul Assay Buffer to Positive Control tube and mix well. Transfer 10ul of the reconstituted Positive Control into separate wells
Note: (1). For unknown samples, perform several dilutions to ensure that catalase activity is within the linear range 0.2 to 5 U/L. (2) The provided catalase serves as a positive control to ensure assay is working and should not be used to calculate the Sample catalase activity
2. Enzyme Reaction. Mix 5ul 3% H2O2 and 914ul dH2O (final 4.8mM). Prepare enough 50uM H2O2 Substrate for sample, positive control and sample blank by mixing, for each well, 1ul of the 4.8mM H2O2 with 95ul Assay Buffer. Note: diluted H2O2 is not stable. Prepare fresh dilutions for each experiment. Add 90ul of the 50uM Substrate to these wells to initiate the catalase reaction. Tap plate quick to mix. Incubate 30 min at room temperature. During the incubation time, proceed with Steps 3 and 4 below
3. H2O2 Standard Curve. Mix 40ul of the 4.8mM H2O2 with 440ul dH2O to yield 400uM H2O2. Prepare standards as shown in the Table below. Transfer 10ul standards into separate wells of the 96-well plate. Add 90ul Assay Buffer to the standards
No 400uM H2O2+H2O Vol (ul) H2O2 (uM)1 100ul+0ul 100 4002 60ul+40ul 100 2403 30ul+70ul 100 1204 0ul+100ul 100 04. Detection. Prepare enough Detection Reagent by mixing, for each reaction well (Sample, Control and Standard wells), 102ul Assay Buffer, 1ul Dye Reagent and 1ul HRP Enzyme. At the end of the 30 min incubation (Step 2), add 100ul Detection Reagent per well. Tap plate to mix. Incubate for 10 min
5. Read optical density at 570nm (550 to 585nm) or fluorescence intensity at lem/ex=585/530nm.
Calculation:Subtract blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the catalase activity of Sample,Catalase (U/L) =RSample Blank–RSampleSlope (uM-1) x 30 min× nRSAMPLE Blank and RSAMPLE are optical density or fluorescence intensity readings of the Sample Blank and Sample, respectively. Slope is determined from the standard curve. 30 min is the catalase reaction time. n is the sample dilution factor
Unit definition: one unit is the amount of catalase that decomposes 1uMole of H2O2 per min at pH 7.0 and room temperature.
Materials Required, But Not Provided:Pipetting devices, centrifuge tubes, uncoated 96-well plates, optical density plate reader, fluorescence plate reader, homogenizer etc.
