Hydrogen peroxide enzyme detection kit

Cat. No.: H1492128

GRADE & PURITY BioReagent

Overview

  This kit enables the colorimetric detection of catalase activity in cells, tissues, and other biological samples. Under conditions of sufficient hydrogen peroxide (H₂O₂) supply, catalase catalyzes the decomposition of H₂O₂ into water and oxygen. The residual H₂O₂ oxidizes a chromogenic substrate in the presence of peroxidase to produce a red product (N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine) with a maximum absorbance at 520 nm. By generating a standard curve using H₂O₂ standards, the amount of H₂O₂ decomposed per unit time and volume can be quantified, allowing calculation of catalase activity in the sample. The kit exhibits high sensitivity, capable of detecting catalase levels as low as 1 U/mL.

  Catalase is widely distributed in living organisms. It is highly abundant in the liver, kidneys, and red blood cells, where it plays a major role in clearing H₂O₂ to prevent oxidative damage.

This kit is suitable for measuring catalase activity in whole blood, erythrocyte lysates, serum, tissue homogenates, cell lysates, and other biological samples.

  One kit provides sufficient reagents for 100 assays.

H1492128	Component	100T	Storage
H1492128A	Assay Buffer	70 mL	2-8℃
H1492128B	H₂O₂ (~1 M)	5 mL	2-8℃
H1492128C	Stop Solution	50 mL	2-8℃. Store in the dark.
H1492128D	Chromogenic Substrate	1 bottle	2-8℃. Store in the dark.
H1492128E	Substrate Buffer	20 mL	2-8℃. Store in the dark.
H1492128F	Peroxidase	1 tube	2-8℃. Store in the dark.

Protocol

1. Reagent Preparation
1.1 Peroxidase Solution: Dissolve one tube of peroxidase in 1.45 mL Assay Buffer. Aliquot and store at -20°C.
1.2 Chromogenic Substrate Solution: Add 20 mL Substrate Buffer to the bottle of chromogenic substrate and mix to dissolve. Aliquot and store at -20°C.
1.3 Preparation of 250 mM H₂O₂: The provided H₂O₂ has a nominal concentration of ~1 M. Due to instability, accurately determine its actual concentration spectrophotometrically before use. Dilute 10 µL of the ~1 M H₂O₂ solution with 990 µL Assay Buffer (~10 mM). Measure A₂₄₀ using a UV spectrophotometer (1 cm pathlength).
H₂O₂ concentration (mM) = 22.94 × A₂₄₀
Calculate the actual concentration of the stock H₂O₂, then prepare a 250 mM H₂O₂ solution using Assay Buffer.
1.4 Preparation of 5 mM H₂O₂: Dilute the accurately measured H₂O₂ stock to 5 mM using Assay Buffer.
1.5 Chromogenic Working Solution: Thaw the Chromogenic Substrate Solution on ice and aliquot to avoid repeated freeze-thaw cycles. Keep other reagents on ice. Dilute Peroxidase Solution 1:1000 in Chromogenic Substrate Solution to prepare the working solution (e.g., add 5 µL Peroxidase Solution to 5 mL Chromogenic Substrate Solution). Mix well.

2. Sample Preparation
When using lysis buffers containing detergents (e.g., Western & IP Lysis Buffer) or hypotonic buffers, dilute samples with the provided Assay Buffer. For samples in isotonic buffers (to maintain peroxisome integrity), use Assay Buffer containing 0.1% Triton X-100. For samples with very low protein concentration (<0.025 mg/mL), add BSA to the buffer to a final concentration of 0.5 mg/mL to stabilize the enzyme.
Tissue extracts exhibit variable catalase activity (highest in liver and kidney, lowest in connective tissue). Cell lysates can typically be used directly. Blood lysates show high activity and require dilution. Refer to the table below for guidance.
2.1 Cell Samples: Lyse cells using Western & IP Lysis Buffer (refer to product instructions).
2.2 Tissue Samples: Lyse tissues using Western & IP Lysis Buffer (refer to product instructions).
2.3 Whole Blood: Collect blood into an anticoagulant tube and mix by inversion. Freeze-thaw 100 µL of whole blood once, then dilute 1000-fold with Assay Buffer before assay.
2.4 Erythrocyte Lysate: Collect blood into an anticoagulant tube and mix. Centrifuge at least 500 µL at 2500 g for 5 min at 4°C. Discard supernatant and wash the pellet three times with ice-cold saline (0.9% NaCl). Resuspend the pellet in ~5 volumes of ice-cold deionized water (e.g., Milli-Q) and incubate on ice for 10 min. Dilute 400-fold with Assay Buffer before use.

Table 1: Sample Usage Guide (Reference Only)

Sample Type	Protein Conc.	Sample Volume	Reaction Time	ΔA₅₂₀ (Blank - Sample)
Blank Control	-	-	-	1.20–1.32 (Initial)
Human Erythrocyte Lysate	0.2 mg/mL	2–6 µL	2 min	0.21–0.71
Mouse Liver Lysate	0.3 mg/mL	5–10 µL	1 min	0.36–0.78
Mouse Kidney Lysate	0.3 mg/mL	5–10 µL	3 min	0.28–0.57
Mouse Spleen Lysate	0.3 mg/mL	5–10 µL	3 min	0.11–0.21
Mouse Brain Lysate	0.7 mg/mL	10–20 µL	3 min	0.014–0.028
HepG2 Lysate	2.0 mg/mL	2–4 µL	3 min	0.21–0.43
Jurkat Lysate	2.0 mg/mL	2–4 µL	3 min	0.28–0.64
Catalase Standard	10000x diluted	2–4 µL	1 min	0.36–0.71

*Note: The values in this table are for reference only. Actual readings may vary significantly due to differences in experimental systems. For uncharacterized samples, try 1, 10, 20, and 50-fold dilutions, use 10 µL, and react for 1 min. Optimal dilution should result in 30–50% consumption of H₂O₂ in 1–5 min for accurate results.*

3. Standard Curve
3.1 Add 0, 12.5, 25, 50, or 75 µL of the prepared 5 mM H₂O₂ solution to 1.5 mL or 0.5 mL microcentrifuge tubes. Adjust the volume to 100 µL with Assay Buffer and mix.
3.2 Transfer 4 µL of each standard to a 96-well plate. Add 200 µL Chromogenic Working Solution. Incubate at 25°C for at least 15 min (not exceeding 45 min) and measure A₅₂₀.
Note: H₂O₂ standards must be prepared fresh daily. This step can be performed concurrently with step 4.6.

4. Sample Assay
Table 2: Reaction Setup

Component	Blank	Sample
Sample Volume	0 µL	X µL
Assay Buffer	40 µL	(40 - X) µL
250 mM H₂O₂	10 µL	10 µL

4.1 Pipette X µL (0–40 µL) of sample into a 1.5 mL tube. Add Assay Buffer to a total volume of 40 µL (i.e., add (40 - X) µL Buffer). Mix gently.
4.2 Add 10 µL of 250 mM H₂O₂ solution and mix quickly. Incubate at 25°C for 1–5 min (refer to Table 1).
4.3 Add 450 µL Stop Solution to terminate the reaction. Mix thoroughly by inversion or vortexing. Proceed to steps 4.4 and 4.5 within 15 min.
4.4 In a new tube, add 40 µL Assay Buffer and 10 µL of the terminated reaction mixture from step 4.3. Mix.
4.5 Transfer 10 µL from the 50 µL mixture in step 4.4 to a well of a 96-well plate. Add 200 µL Chromogenic Working Solution.
4.6 Incubate at 25°C for at least 15 min (not exceeding 45 min) and measure A₅₂₀. 

5. Calculation of Catalase Activity
5.1 Generate standard curve: A₅₂₀ = k × [H₂O₂ (µM)] + b. Determine k and b.
5.2 Calculate residual H₂O₂ in sample: [Residual H₂O₂ (µM)] = (A₅₂₀ - b) / k
5.3 Unit Definition: One unit (U) of catalase activity is defined as the amount of enzyme that decomposes 1 µM of H₂O₂ per minute at 25°C and pH 7.0.
5.4 For cell or tissue samples:
[Catalase Activity] = [ΔH₂O₂ (µM)] × [Dilution Factor] / ([Reaction Time (min)] × [Sample Volume (mL)] × [Protein Concentration (mg/mL)])
Units: U/mg protein
[ΔH₂O₂ (µM)] = [Residual H₂O₂ in Blank (µM)] - [Residual H₂O₂ in Sample (µM)]
[Dilution Factor] = 250
[Reaction Time] = actual reaction time in minutes
[Sample Volume] = X µL from Table 2, expressed in mL (X/1000 mL)
[Protein Concentration] = protein concentration of the sample (mg/mL) when X µL was used.
5.5 For liquid samples (e.g., plasma):
[Catalase Activity] = [ΔH₂O₂ (µM)] × [Dilution Factor] / ([Reaction Time (min)] × [Sample Volume (mL)])
Units: U/mL sample

Precautions

1. Catalase samples (pure enzyme or lysates) can be stored at 4°C for ~1 week or at -70°C for long-term storage. Avoid -20°C, as it significantly reduces activity.

2. The chromogenic reaction (step 4.5) must be initiated within 15 min after adding Stop Solution.

3. H₂O₂ is unstable. Determine its concentration accurately as described before use.

4. For precise quantification of catalase activity, a protein assay kit (e.g., BCA Protein Assay Kit) is required to determine sample protein concentration.

5. Wear appropriate personal protective equipment (lab coat, gloves) during reagent handling.

6. For research use only.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.