Catalase Fluorescent Activity Kit

Catalog No. ABIN2866577

Product Details

Target: Catalase (CAT)

Reactivity: Human, Various Species

Detection Method: Fluorometric

Application: Activity Assay (AcA)

Purpose: The DetectX® Catalase Activity Kit is designed to quantitatively measure catalase activity in a variety of samples.

Brand: DetectX®

Sample Type: Serum, Plasma, Cell Samples, Tissue Samples, Cell Lysate

Components: Black 96 well Half Area Plates 2 Plates
Catalase Standard 90 μL 100 Unit/mL of bovine catalase in a special solution.
Assay Buffer Concentrate 25 mL A 5X buffer concentrate containing detergents and stabilizers.
Hydrogen Peroxide Reagent 5 mL Hydrogen peroxide solution containing stabilizers.
Fluorescent Detection Reagent 5 mL A solution of the substrate in a special stabilizing buffer.
Horseradish Peroxidase Concentrate 60 μL A 100X concentrated solution of HRP in a special stabilizing solution.

Material not included: Repeater pipet with disposable tips capable of dispensing 25 μL. 96 well plate reader capable of reading fluorescence at 580-590 nm with excitation at 570-580 nm.
Set plate parameters for a 96-well Corning Costar 3694 plate.
See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
Software for converting fluorescent intensity readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Protocol: A bovine catalase standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve.
Samples are diluted in the provided Assay Buffer and added to the wells of a half area black plate.
Hydrogen peroxide is added to each well and the plate incu- bated at room temperature for 30 minutes.
The supplied Fluorescent Detection Reagent is added, followed by diluted horseradish peroxidase and incubated at room temperature for 15 minutes.
The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a fluorescent product.
The fluorescent product is read at 590 nm with excitation at 570 nm.
Increasing levels of catalase in the samples causes a decrease in H2O2 concentration and a reduction in fluorescent product.
The activity of the catalase in the sample is calculated after making a suitable correction for any dilution, using software available with most plate readers.
The results are expressed in terms of units of catalase activity per mL.

Reagent Preparation:

Vortex the suspension of HRP prior to pipetting.
Pipet from the base of the tube. 1/2 Plate 1 Plate 1.5 Plates 2 Plates Horseradish Peroxidase 15 μL 25 μL 38 μL 50 μL Assay Buffer 1.485 mL 2.475 mL 3.762 mL 4.95 mL Final Mixture 1.5 mL 2.5 mL 3.8 mL 5 mL The HRP Preparation will be stable for one day.

Sample Preparation:

Cell Suspensions and Adherent Cells 1. Centrifuge > 1 x 106 cells in suspension at 250 x g for 10 minutes at 4 °C. Discard the supernatant. Adherent cells should be gently dislodged using a rubber policeman - do not use proteolytic enzymes. 2. Homogenize or sonicate the pellet in 1-2 mL of cold Assay Buffer per 100 mg of cells. Centrifuge at 10,000 x g for 15 minutes at 4 °C. 3. Collect the supernatant and assay immediately, or store at ≤-70 °C. Dilute in Assay Buffer prior to measuring catalase activity.

Assay Procedure:

Use the plate layout sheet on the back page to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3694 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
1. Pipet 25 μL of samples or appropriate standards into duplicate wells in the plate.
2. Pipet 25 μL of Assay Buffer into duplicate wells as the Zero standard.
3. Add 25 μL of the supplied Hydrogen Peroxide Reagent to each well using a repeater pipet.
4. Incubate at room temperature for 30 minutes.
5. Add 25 μL of the supplied Fluorescent Detection Reagent to each well using a repeater pipet.
6. Initiate the reaction by adding 25 μL of the prepared HRP Reagent to each well using a repeater pipet.
7. Incubate at room temperature for 15 minutes.
8. Set plate parameters for a 96-well Corning Costar 3694 plate. Read the fluorescent emission at 585 ± 5 nm with excitation at 575 ± 5 nm. Please contact your plate reader manufacturer for suitable filter sets.

Calculation of Results:

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader.
The sample activity obtained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from
Sample Mean FLU Catalase Activity (U/mL) Standard 1 1,658 5.0 Standard 2 6,276 2.5 Standard 3 15,513 1.25 Standard 4 23,794 0.625 Standard 5 29,495 0.313 Standard 6 32,824 0.156 Zero 35,198 0 Sample 1 5,440 2.88 Sample 2 25,092 0.55 Always run your own standard curves for calculation of results.
Do not use these data.
Catalase Unit Definition One Unit of Catalase decomposes one micromole of H2O2 per minute at 25 °C and pH 7.0.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The supplied hydrogen peroxide solution contains very dilute H2O2.
Sample typeS and preparatiOn Samples that need to be stored after collection should be stored at -70°C or lower, preferably after being frozen in liquid nitrogen.
This assay has been validated for serum, plasma and erythrocyte lysates.
Samples containing visible particulate should be centrifuged prior to using.
Process any cell pellet as described for Cell Lysates.

Storage: 4 °C,RT

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit.

References

Walliczek-Dworschak, Schöps, Feron, Brignot, Hähner, Hummel: "Differences in the Density of Fungiform Papillae and Composition of Saliva in Patients With Taste Disorders Compared to Healthy Controls." in: Chemical senses, Vol. 42, Issue 8, pp. 699-708, (2017) (PubMed).

Mapanga, Joseph, Symington, Garson, Kimar, Kelly-Laubscher, Essop: "Detrimental effects of acute hyperglycaemia on the rat heart." in: Acta physiologica (Oxford, England), Vol. 210, Issue 3, pp. 546-64, (2014) (PubMed).

Target Details

Target: Catalase (CAT)

Alternative Name: Catalase (CAT Products)

Synonyms: 2210418N07 Kit, Cas-1 Kit, Cas1 Kit, Cs-1 Kit, CS1 Kit, Cat01 Kit, Catl Kit, fb68a12 Kit, wu:fb68a12 Kit, CAT Kit, CATA Kit, CG6871 Kit, CT21282 Kit, CatA Kit, DMCATHPO Kit, DROCATHPO Kit, Dmel\\CG6871 Kit, U00145 Kit, bs36h11.y1 Kit, cat Kit, GB11648 Kit, catalase Kit, Cat Kit, BA0843 Kit, DKFZp469E0232 Kit, catalase Kit, Catalase Kit, catalase, gene 2 Kit, CAT Kit, Cat Kit, cat Kit, cat.2 Kit, LOC769804 Kit, BA_0843 Kit

Background: Hydrogen peroxide, H2O2 is one of the most frequently occurring reactive oxygen species. It is formed either in the environment or as a by-product of aerobic metabolism, superoxide formation and dismutation, or as a product of oxidase activity. Both excessive hydrogen peroxide and its decomposition product hydroxyl radical, formed in a Fenton-type reaction, are harmful for most cell components. Its rapid removal is essential for all aerobically living prokaryotic and eukaryotic cells1,2. Hydrogen peroxide however can act as a second messenger in signal transduction path- ways, in immune cell activation, inflammation processes, cell proliferation, and apoptosis3-5. Catalase Catalase Reaction 2 H2O2 ➞ H2O + O2 One of the most efficient ways of removing peroxide is through the enzyme catalase, which is encoded by a single gene, and is highly conserved among species6-8. Mammals, including humans and mice, express catalase in all tissues, and a high concentration of catalase can be found in the liver, kidneys and erythrocytes9,10. The expression is regulated at transcription, post-transcription and post-translation levels6, 11. High catalase activity is detected in peroxisomes12. More recent- ly, short wavelength UV radiation has been shown to produce reactive oxygen species (ROS) through the action of catalase13. This response is thought to act as a mechanism to protect DNA by converting damaging UV radiation into ROS species that can be metabolized and detoxified by cellular antioxidant enzymes