OxiSelect™ Catalase Activity Assay Kit, Fluorometric

Catalog No. ABIN2344993

Product Details

Reactivity: Others

Detection Method: Fluorometric

Application: Activity Assay (AcA)

Purpose: The OxiSelect™ Catalase Activity Assay Kit is a sensitive quantitative fluorometric assay for detecting catalase activity.

Brand: OxiSelect™

Sample Type: Cell Lysate, Serum, Plasma

Analytical Method: Quantitative

Sensitivity: 30 mU/mL

Characteristics: OxiSelect™ Catalase Activity Assay Kit is a quantitative fluorometric assay for measuring catalase activity from biological samples. The kit employs a simple, sensitive, homogeneous no wash and HTS-compatible assay for measuring catalase activity in samples such as cell lysates, plasma, and tissue homogenates. Catalase-containing samples can be incubated in a known amount of hydrogen peroxide. In the presence of H2O2 and horseradish peroxidase (HRP), non-fluorescent ADHP (10-Acetyl- 3, 7-dihydroxyphenoxazine) is oxidized to the highly fluorescent Resorufin. ADHP is known as one of the most sensitive and stable fluorogenic probes for detecting H2O2. The probe can be utilized to monitor H2O2 release from cells or enzyme-coupled reactions. The kit has a detection sensitivity limit of at least 30 mU/mL catalase. Each kit provides sufficient reagents to perform up to 500 assays, including standard curve and unknown samples.

Components:

1. ADHP Probe : One 250 μL amber tube of a 10 mM solution in DMSO.
2. HRP : One 100 μL tube of a 100 U/mL solution in glycerol*.
3. Hydrogen Peroxide : One 100 μL amber tube of an 8.82 M solution.
4. 10X Assay Buffer : One 25 mL bottle.
5. Catalase Standard : One 50 μL amber tube of 600,000 Units/mL**. *Note: One unit is defined as the amount of enzyme that will form 1.0 mg purpurogallin from pyrogallol in 20 seconds at pH 6.0 and 20°C. **Note: One unit is defined as the amount of enzyme that will decompose 1.0 μmole of hydrogen peroxide per minute at pH 7.0 and 25°C.

Material not included:

1. Distilled or deionized water
2. 1X PBS for sample dilutions
3. 37 °C incubator
4. Multichannel micropipette reservoir
5. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
6. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
7. Standard 96-well fluorescence black microtiter plate and/or black cell culture microplate 3
8. Fluorescence microplate reader capable of reading excitation in the 530-570 nm range and emission in the 590-600 nm range.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Quantify catalase activity in about 60 minutes
• Suitable for use with whole blood, plasma, serum, cell lysates or tissue homogenates
• Catalase standard included for absolute quantitation

Protocol: First, catalase induces decomposition of H2O2 to produce water and oxygen (O2). The rate of disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of catalase (See Reaction 1 in . Next, in the presence of HRP, ADHP reacts with the remaining H2O2 in a 1:1 stoichiometry to produce highly fluorescent Resorufin. As the catalase activity increases, the Resorufin signal decreases. The Resorufin product can be easily read by a fluorescence microplate reader with an excitation filter of 530-560 nm and an emission filter of ~590 nm (See Reaction 2 in . Fluorescence values are proportional to the catalase levels within the samples. The catalase content in unknown samples is determined by comparison with the predetermined catalase standard curve. 2 Catalase Reaction 1: 2 H2O2 2 H2O + O2 Reaction 2: H O (Remaining) + ADHP HRP 2 2 Resorufin (Fluorescence) .

Reagent Preparation:

Note: All reagents must be brought to room temperature prior to use.

• 1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution. Stir or vortex to homogeneity.
• ADHP/HRP Working Solution: Prepare an ADHP/HRP Working Solution by adding ADHP to a final concentration of 100 μM and HRP to a final concentration of 0.4 U/mL in 1X Assay Buffer (eg. Add 50 μL ADHP stock solution and 20 μL HRP stock solution to 4.930 mL 1X Assay Buffer). This volume is enough for ~100 assays. The ADHP/HRP Working Solution is stable for 1 day. Prepare only enough for immediate applications. Protect the Working Solution from light.
• Hydrogen Peroxide Working Solution: Prepare a Hydrogen Peroxide Working Solution by diluting the stock concentration to 40 μM in 1X Assay Diluent. To prepare the H2O2, first perform a 1:1000 dilution of the stock H2O2 in deionized water. Use only enough for immediate applications (eg. Add 5 μL of H2O2 to 4.995 mL deionized water). This solution has a concentration of 8.82 mM (8820 μM). Use this 8.82 mM H2O2 solution to prepare a 40 μM H2O2 solution in 1X Assay Buffer (eg. Add 12.5 μL of the prepared 8.82 mM H2O2 solution to 2.75 mL 1X Assay Buffer). Mix thoroughly. This volume is enough for ~100 assays. The Hydrogen Peroxide Working Solution is stable for 1 day. Prepare only enough for immediate use.

Sample Preparation:

All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator.

• Cell Culture Supernatant: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Prepare the catalase standard curve in the same non-conditioned media. Serum samples are not recommended since serum could possibly interfere with the assay. Note: Maintain pH between 7 and 8 for optimal working conditions as the ADHP is unstable at high pH (>8.5).
• Cell Lysates: Resuspend cells at 1-2 x 106 cells/mL or tissues at 50 mg/mL in 1X Assay Buffer or PBS. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. The lysate can be assayed undiluted or titrated in 1X Assay Buffer or PBS.
• Plasma or Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted in 1X Assay Buffer or PBS. 4 Notes:
• All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.
• A serial dilution will be necessary depending on the total H2O2 present. Extremely high levels of H2O2 (≥ 500 μM final concentration) can lower the fluorescence because excess H2O2 can further oxidize the reaction product, Resorufin, to nonfluorescent product Resazurin.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the ADHP probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL (Votyakova and Reynolds, Ref. 3).
• Avoid samples containing DTT or β-mercaptoethanol since Resorufin is not stable in the presense of thiols (above 10 μM).

Assay Procedure:

1. Prepare and mix all reagents thoroughly before use. Each cell sample, including unknown and standard, should be assayed in duplicate or triplicate.
2. Add 25 μL of catalase standards, controls, and samples into individual microtiter plate wells.
3. Add 25 μL of the 40 μM Hydrogen Working Solution into each microplate well containing standards, samples and controls. Mix thoroughly.
4. Incubate the reaction mixture for 30 minutes at room temperature.
5. Add 50 μL of the ADHP/HRP Working Solution to each well with standards, controls, and samples. Mix the well contents thoroughly and incubate on a shaker for 30 minutes at 37 °C and protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
6. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.
7. Calculate the activity of catalase within samples by comparing the sample absorbance to the standard curve.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt, store the ADHP probe and HRP at -20°C. ADHP is light sensitive and must be stored accordingly. Avoid multiple freeze/thaw cycles. Store the remaining kit components at 4°C.

References

Mels, Huisman, Smith, Schutte, Schwedhelm, Atzler, Böger, Ware, Schutte: "The relationship of nitric oxide synthesis capacity, oxidative stress, and albumin-to-creatinine ratio in black and white men: the SABPA study." in: Age (Dordrecht, Netherlands), Vol. 38, Issue 1, pp. 9, (2016) (PubMed).

Iqbal, Riaz, Andrabi, Shahzad, Durrani, Ahmad: "l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa." in: Andrologia, (2016) (PubMed).

van Zyl, Huisman, Mels: "Antioxidant enzyme activity is associated with blood pressure and carotid intima media thickness in black men and women: The SABPA study." in: Atherosclerosis, Vol. 248, pp. 91-6, (2016) (PubMed).

du Plooy, Martha Cornelia Mels, Huisman, Kruger: "The association of endothelin-1 with markers of oxidative stress in a biethnic South African cohort: the SABPA study." in: Hypertension research : official journal of the Japanese Society of Hypertension, Vol. 40, Issue 2, pp. 189-195, (2016) (PubMed).

Target Details

Background: Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species (ROS) and antioxidants. However, excessive ROS accumulation will lead to cellular injury, such as damage to DNA, proteins, and lipid membranes. The cellular damage caused by ROS has been implicated in the development of many disease states, such as cancer, diabetes, cardiovascular disease, atherosclerosis, and neurodegenerative diseases. Under normal physiological conditions, cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes and other redox molecules. Because of their potential harmful effects, excessive ROS must be promptly eliminated from the cells by this variety of antioxidant defense mechanisms. Hydrogen peroxide is an ROS that is a toxic product of normal aerobic metabolism and pathogenic ROS production involving oxidase and superoxide dismutase reactions. Hydrogen peroxide is poisonous to eukaryotic cells and in high doses can initiate oxidation of DNA, lipids, and proteins, which can lead to mutagenesis and cell death. The cellular damage caused by H2O2 has been implicated in the development of many pathological conditions such as aging, asthma, arthritis, diabetes, cardiovascular disease, atherosclerosis, Down's Syndrome, and neurodegenerative diseases. Catalase is an antioxidant enzyme omnipresent in mammalian and non-mammalian cells that destroys hydrogen peroxide by dismutation. Eukaryotic catalases are heme enzymes found in the liver, kidney, and erythrocytes in high concentrations while the lowest concentrations are in the connective tissues. The enzyme is concentrated in the peroxisome subcellular organelles.