Catalase (CAT), also known as hydroperoxidase, is a conjugated enzyme with an iron porphyrin prosthetic group. It is a tetrameric enzyme composed of four identical subunits, containing four molecules of heme as prosthetic groups, with a molecular weight of approximately 24 kD. CAT rapidly scavenges hydrogen peroxide, a toxic substance produced by cellular metabolism, and works together with GSH-Px to protect sulfhydryl enzymes, membrane proteins, and facilitate hydrogen peroxide decomposition.
Assay Principle
Under optimal enzymatic reaction conditions, the residual H₂O₂ in samples like serum or plasma reacts with ammonium molybdate to form a stable yellow complex. The intensity of the yellow color is inversely proportional to the enzyme activity. The absorbance at 405 nm is measured using a microplate reader. The detection of this enzyme holds value for researching the balance of free radical metabolism, anti-aging mechanisms, and the pathogenesis of tumors. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
C1505488
| Component
| 100T | Storage
|
C1505488A
| H₂O₂ Stock Solution
| 1 mL×2 | 2-8℃. Store in the dark.
|
C1505488B
| CAT Assay Buffer | 100 mL | RT. |
C1505488C
| Ammonium Molybdate Reagent
| 1 g | RT. |
Required Materials Not Provided
1. Distilled water, Physiological saline
2. Mortar and pestle or homogenizer, Centrifuge tubes, Centrifuge, Water bath or incubator, Microplate reader, Spectrophotometer, 96-well plate
Experimental Procedure
1. Preparation of 65 mM H₂O₂ Substrate Solution
The provided H₂O₂ Stock Solution is approximately 1 M. As H₂O₂ is not very stable, determine its actual concentration before use.
Dilute the ~1 M H₂O₂ Stock Solution 100-fold with CAT Assay Buffer to achieve ~10 mM.
Measure the absorbance at 240 nm (A240) using a spectrophotometer.
Calculate the H₂O₂ concentration using the formula: H₂O₂ concentration (mM) = A240 × 22.94.
Using this calculated actual concentration, prepare the 65 mM H₂O₂ Substrate Solution by appropriate dilution with CAT Assay Buffer.
2. Preparation of MO Chromogenic Solution
Weigh 0.4 g of Ammonium Molybdate Reagent and add it to 10 mL of distilled water. Dissolve completely.
Note: The MO Chromogenic Solution may develop a milky-white precipitate; if so, discard it. Prepare fresh before use. The provided Ammonium Molybdate Reagent is supplied in excess.
3. Sample Preparation
3.1 Cell or Tissue Samples
Lyse an appropriate amount of cells or tissue using RIPA Lysis Buffer. Homogenize if necessary.
Centrifuge at low speed and collect the supernatant.
Store at -20°C for CAT detection.
3.2 Plasma, Serum, and Urine Samples
Prepare plasma and serum by conventional methods. Dilute 10-fold with physiological saline before direct assay.
Urine can usually be assayed directly.
Store at -20°C if not assayed immediately.
3.3 Whole Blood Samples
Collect an appropriate volume of whole blood into an anticoagulant tube and mix by inverting.
Freeze-thaw the whole blood once.
Dilute 1000-fold with CAT Assay Buffer before CAT assay.
3.4 Erythrocyte Lysate from Blood
Collect blood in an anticoagulant tube and mix.
Take at least 500 µL whole blood, centrifuge at 3000 g for 5 min at 4°C, discard supernatant.
Wash the pellet 3 times with ice-cold physiological saline.
Resuspend the cell pellet in approximately 5 volumes of ice-cold deionized water (e.g., Milli-Q water).
Incubate on ice for 10 minutes.
Dilute 400-fold with CAT Assay Buffer before CAT assay.
3.5 Plant Samples
Precisely weigh 0.5 g of plant material (pulp or leaves without veins), mince, and place in a pre-chilled (4°C) mortar or homogenizer.
Add 1 mL of pre-chilled Extraction Buffer and grind at low temperature to homogenate.
Transfer to a centrifuge tube, rinse the mortar/homogenizer with 3 mL Extraction Buffer, and combine into the tube.
Adjust the final volume to 5 mL with Extraction Buffer.
Centrifuge at 10,000 rpm/min for 20 min at 4°C. The supernatant is the enzyme extract for CAT assay.
Extraction Buffer Recipe: Dissolve 3.3 g anhydrous Disodium Hydrogen Phosphate and 0.13 g Sodium Dihydrogen Phosphate Dihydrate in water, bring final volume to 200 mL. Store at 4°C.
Note: If CAT activity is low, reduce the total volume of Extraction Buffer to increase the enzyme concentration.
3.6 High-Activity Samples
If the sample contains high CAT activity, dilute it with CAT Assay Buffer.
3.7 (Optional) Protein Concentration Assay
After sample preparation, determine the protein concentration using a BCA Protein Assay Kit to calculate CAT content per mg protein. It is recommended to use Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-use BCA Protein Quantification Kit.
4. CAT Assay Setup
Set up Blank, Self-Control, and Test tubes according to the table below.
Add reagents in the specified order, avoiding bubbles.
For high enzyme activity, reduce sample volume or dilute before assay. Duplicate tubes are recommended.
Reagent (µL)
| Blank Tube
| Self-Control Tube
| Test Tube
|
CAT Assay Buffer
| 10
| —
| —
|
Sample
| —
| —
| 10
|
65 mM H₂O₂ Substrate Solution (pre-warmed 5min, 37°C)
| 100
| 100
| 100 |
Mix immediately and incubate at 37°C for EXACTLY 60 seconds.
Sample
| —
| 10
| —
|
MO Chromogenic Solution
| 100
| 100
| 100 |
5. CAT Measurement
Zero the instrument with distilled water.
Measure the absorbance at 405 nm using a microplate reader.
Record the absorbance for each tube (ABlank, ASelf-Control, ATest).
Note: For greater accuracy, use a spectrophotometer if available.
6. Calculation of Results
Definition of CAT Activity Unit: One unit of CAT enzyme activity is defined as the amount of enzyme that catalyzes the decomposition of 1 μmol of H₂O₂ per minute at 37°C.
6.1 CAT Activity in Serum, Plasma, Urine:
CAT Activity (U/mL) = { (ASelf-Control - ATest) / ABlank } × 650 × N
6.2 CAT Activity in Tissues, Cells:
CAT Activity (U/mg prot) = { (ASelf-Control - ATest) / ABlank } × 650 / Cpr
(Where Cpr is the sample protein concentration in mg/mL)
6.3 CAT Activity in Plant Tissues:
CAT Activity (U/g weight) = { (ASelf-Control - ATest) / ABlank } × 650 × N × V / m
Parameter Definitions:
ASelf-Control: Absorbance of Self-Control tube
ATest: Absorbance of Test tube
ABlank: Absorbance of Blank tube
N: Dilution factor of the test sample before assay
V: Total volume of extraction buffer (mL)
m: Mass of plant sample (g) = 0.5 (as per protocol)
Precautions
1. This kit can also be used with a spectrophotometer, but the number of samples processed will be correspondingly reduced.
2. The test sample should not contain CAT inhibitors, and repeated freeze-thaw cycles should be avoided.
3. If the CAT Assay Buffer becomes turbid or develops flocculent matter, discard it.
4. The MO Chromogenic Solution is stable for 2 weeks at 4°C after preparation. Discard if a milky-white precipitate forms; prepare fresh is best.
5. For plant samples, grind rapidly to prevent loss of CAT activity, and keep samples on ice during processing. This method is not recommended for plant samples; use Catalase Activity Assay Kit (Ultraviolet Absorption Method) is preferred.
6. CAT in intact red blood cells and undiluted hemolysate is relatively stable for 1 week at 4°C. CAT in diluted hemolysate loses activity easily.
7. Avoid hemolysis caused by sample freezing, as it can decrease catalase activity by 10-15%.
8. Serum CAT activity decreases 64.7% at room temperature in 3 days, 10.5% at 4°C, but only 3.5% after 30 days at -20°C. Store test samples at -20°C or -70°C.
9. For your safety and health, wear lab coats and disposable gloves during operation.
10.Use reagents promptly after opening to avoid affecting subsequent experimental results.
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