Ascorbate Peroxidase (APX) Activity Assay Kit

Ascorbate Peroxidase (APX) Activity Assay Kit

SKU #	E-BC-K353-S
Detection Instrument	Spectrophotometer (290 nm)
Detection method	Colorimetric method

Product Details

Properties

Synonyms	APX
Sample type	Plant tissue
Sensitivity	0.071 U/g tissue
Detection range	0.071-47 U/g tissue
Detection Method	Colorimetric method
Assay type	Enzyme Activity
Assay time	60 min
Precision	Average inter-assay CV: 6.400% | Average intra-assay CV: 4.800%
Other instruments required	Vortex mixer, Micropipettor, Water bath, Incubator, Centrifuge
Other reagents required	Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4)
Storage	2-8℃
Valid period	12 months

Images

H Y Lee et al investigate the effects of melatonin in high light stress tolerance in Arabidopsis thaliana. Ascorbate peroxidase (APX) activity of Arabidopsis leaf was determined using APX activity assay kit (E-BC-K353-S).

In high light stress situation, the APX activity was significantly reduced in snat1 mutant group. (P<0.05)

 

Dilution of Sample

It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range (0.071-47 U/g tissue).

The recommended dilution factor for different samples is as follows (for reference only):

Sample type	Dilution factor
10% Epipremnum aureum tissue homogenization	1
10% Carrot tissue homogenization	1
10% Green pepper tissue homogenization	1
10% Mushrooms tissue homogenization	1

 

Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

 

Detection Principle

Ascorbate Peroxidase (APX) can catalyze the reaction between ascorbic acid (ASA) and hydrogen peroxide (H2O2), and ASA can be oxidized to monodehydroascorbic acid (MDASA). The absorbance of solution at 290 nm will decline as the oxidation of ASA. The APX activity can be calculated by detecting the decrease of A290.

Kit Components & Storage

Item	Component	Size 1 (50 assays)	Size 2 (100 assays)	Storage
Reagent 1	Extracting Solution	60 mL × 1 vial	60 mL × 2 vials	2-8°C, 12 months
Reagent 2	Buffer Solution	40 mL × 1 vial	40 mL ×2 vials	2-8°C, 12 months
Reagent 3	Substrate	Powder ×1 vial	Powder ×2 vials	2-8°C, 12 months shading light
Reagent 4	Substrate Solution	12 mL ×1 vial	12 mL ×1 vial	2-8°C, 12 months

 

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

 

Technical Data:

Parameter:

Intra-assay Precision

Three human serum samples were assayed in replicates of 20 to determine precision within an assay. (CV = Coefficient of Variation)

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/g tissue)	1.50	18.40	32.80
%CV	5.2	4.7	4.5

Inter-assay Precision

Three human serum samples were assayed 17 times in duplicate by three operators to determine precision between assays.

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/g tissue)	1.50	18.40	32.80
%CV	5.7	6.7	6.8

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 96%.

	Standard 1	Standard 2	Standard 3
Expected Conc. (U/g tissue)	8.5	26.5	40.5
Observed Conc. (U/g tissue)	8.4	24.4	39.3
Recovery rate (%)	99	92	97

 

Sensitivity

The analytical sensitivity of the assay is 0.071 U/g tissue. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.