Catalase Assay Kit

1. 
   

   Introduction:

   
   

Catalase (EC 1.11.1.6) is a ubiquitous antioxidant enzyme that is present in nearly all living organisms. It functions to catalyze the decomposition of hydrogen peroxide (H₂0₂) to water and oxygen. BioVision’s Catalase Assay Kit provides a highly sensitive, simple, direct and HTS-ready assay for measuring Catalase activity in biological samples. In the assay, catalase first reacts with H₂O₂ to produce water and oxygen, the unconverted H₂O₂ reacts with OxiRed™ probe to produce a product, which can be measured at 570 nm (Colorimetric method) or at Ex/Em=535/587nm (fluorometric method). Catalase activity is reversely proportional to the signal. The kit detects high pico-unit of catalase in samples.

2. 
   

   Kit Contents:

   
   

3. 
   

   Storage and Handling:

   
   

Store the kit at -20^oC, protect from light. Warm the assay buffer to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

OxiRed™ Probe: Dissolve each vial with 110 µl DMSO (provided, needs hand-warm up to 18C to become liquid) prior to use; one vial is sufficient for 50 assays.

HRP Solution: Dissolve with 220 l Assay Buffer; it is sufficient for 100 assays.

Positive Control Solution: Dissolve positive control vial into 500 µl Assay Buffer. Aliquot 100 µl per vial, store at -20°C.

Warm the Assay Buffer to room temperature before use. Keep samples, HRP and Catalase on ice during the assay. All these components are stable for 2 week at 4°C, or for 1 month at -20°C after reconstitution.

5. 
   

   Catalase Activity Assay:

   
   

0. 
   

   Sample and Positive Control Preparations:
   
   Homogenize 0.1 gram tissues, or 10⁶ Cells, or 0.2 ml Erythrocytes on ice in 0.4-1.0 ml cold Assay Buffer; Centrifuge at 10,000 x g for 15 min at 4°C; Collect the supernatant for assay, keep on ice. Liquid samples can be tested directly. Store samples at -80°C for storage.

   
   

Add 2-78 µl samples or 1-10 µl Positive Control Solution into each well, and adjust volume to total 78 µl with Assay Buffer. Prepare sample High Control (HC) with the same amount of sample in separate wells; bring total volume to 78 µl with Assay Buffer. Add 10 µl of Stop Solution into the sample High Control, mix and incubate at 25°C for 5 min to completely inhibit the catalase activity in samples as High Control. For unknown samples, we suggest testing several doses of your sample to ensure the readings are within the linear range.

Reducing agents in samples may interfere with the assay. Keep DTT or 2-mercaptoethanol below 5 μM.

1. 
   

   H₂O₂ Standard Curve:

   
   

Dilute 5 µl of 0.88M H₂O₂ into 215 µl DiH₂O to generate 20 mM H₂O₂, then take 50 µl of the 20 mM H₂O₂ and dilute into 0.95 ml DiH₂O to generate 1 mM H₂O₂. Add 0, 2, 4, 6, 8, 10 µl of 1 mM H₂O₂ solution into 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well H₂O₂ standard. Bring the

final volume to 90 µl with Assay Buffer. Add 10 µl Stop Solution into each well. For the fluorometric assay, dilute the standard H₂O₂ 10 more folds for the standard curve. Diluted H₂O₂ is unstable, prepare fresh dilution each time.

2. 
   

   Catalase Reaction:

   
   

Add 12 µl fresh 1 mM H₂O₂ into each well of both samples and sample High Controls to start the reaction, incubate at 25°C for 30 min, and then add 10 µl Stop solution into each sample vial to stop the reaction (Note: High Control and standard curve wells already contain Stop Solution).

3. 
   

   Develop Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Develop Mix:

   
   

46 μl Assay Buffer

2 μl OxiRed™ Probe

2 μl HRP solution

Add 50 μl of the Develop Mix to each test samples, controls, and standards. Mix well and incubate at 25°C for 10 min. Measure O.D. 570 nm in a plate reader.

For low amount catalase, you can either increase the incubate time prior to adding the Stop Solution or use fluorometric method. For the fluorimetric method, decrease the 1 mM H₂O₂ amount to1.5 µl and OxiRed™ Probe to 0.3 µl in the reaction.

4. 
   

   Calculation: Signal changes by catalase in sample is ∆A = A_HC – A_Sample. A_HC is the reading of sample High Control, A_Sample is the reading of sample in 30 min. Plot the H₂O₂ Standard Curve. Apply the ∆A to the H₂O₂ standard curve to get B nmol of H₂O₂ decomposed by catalase in 30 min reaction. Catalase activity can be calculated:

   
   

CAT Activity = Sample Dilution Factor = nmol/min/ml = mU/mL

Where: B is the decomposed H₂O₂ amount from H₂O₂ Standard Curve (in nmol).

V is the pretreated sample volume added into the reaction well (in ml).

30 is reaction time 30 min.

Unit definition: One unit of catalase is the amount of catalase decomposes 1.0 μmol of H₂O₂ per min at pH 4.5 at 25 °C