[E-BC-K096-M] Glutathione Peroxidase (GSH-Px) Activity Assay Kit > Biochemical Assay Kits

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[E-BC-K096-M] Glutathione Peroxidase (GSH-Px) Activity Assay Kit 요약정보 및 구매

96T

상품 선택옵션 0 개, 추가옵션 0 개

제조사	Elabscience
납기일	1-2 주
판매가격	364,000원
포인트	0점(구매금액 : 0%)
배송비결제	3,500원 (주문금액 10만원 이상일시 배송비 무료)

선택된 옵션

[E-BC-K096-M] Glutathione Peroxidase (GSH-Px) Activity Assay Kit (+0원)
수량

이전상품 [E-BC-K096-S] Glutathione Peroxidase (GSH-Px) Activity Assay Kit 다음 상품 [E-BC-K094-S] Acid Phosphatase (ACP) Activity Assay Kit

상품 기본설명

96T

상품 상세설명

General information

Detection significance

Glutathione Peroxidase (GSH-PX) is an important enzyme that catalyzes decomposition of hydrogen peroxide. GSH specifically catalyze the reaction between GSH and hydrogen peroxide, protecting cell membrane structure and keeping membrane function integrity. Se-cysteine is the active center of the GSH-PX. Determination of GSH-PX activity in organism can be an indicator of selenium level as Se is essential section of GSH-PX.

Detection principle

Glutathione Peroxidase (GSH-PX) can promote the reaction of hydrogen peroxide (H2O2) and reduced glutathione to produce H2O and oxidized glutathione (GSSG). The activity of glutathione peroxidase can be expressed by the rate of enzymatic reaction. The activity of glutathione can be calculated by measuring the consumption of reduced glutathione. Hydrogen peroxide (H2O2) and reduced glutathione can react without catalysis of GSH-PX, so the portion of GSH reduction by non-enzymatic reaction should be subtracted. GSH can react with dinitrobenzoic acid to produce 5-thio-dinitrobenzoic acid anion, which showed a stable yellow color. Measure the absorbance at 412nm, and calculate the amount of GSH.

The key point

1. The supernatant after centrifugation after adding reagent 2 in enzymatic reaction must be clarified.

2. Determine optimal dilution factor of samples before formal experiment. It is recommended to choose the optimal dilution factor when inhibition ratio in the range of 20%~30%.

3. Reagent 1 application solution should be preheated in advance at 37℃

Operation procedures

Plate set up

S1'

S9'

S17

S17'

S25

S25'

S33

S33'

S2'

S10

S10'

S18

S18'

S26

S26'

S34

S34'

S3'

S11

S11'

S19

S19'

S27

S27'

S35

S35'

S4'

S12

S12'

S20

S20'

S28

S28'

S36

S36'

S5'

S13

S13'

S21

S21'

S29

S29'

S37

S37'

S6'

S14

S14'

S22

S22'

S30

S30'

S38

S38'

S7'

S15

S15'

S23

S23'

S31

S31'

S39

S39'

S8'

S16

S16'

S24

S24'

S32

S32'

S40

S40'

[Note]: A-H, standard wells; S1-S40：Non-enzyme wells; S1'-S40', Enzyme wells.

The dilution of standard curve

Dilute 100 μmol/L GSH standard solution with GSH standard application solution to a serial concentration. The recommended dilution gradient is as follows: 100, 80, 60, 50, 40, 20, 10, 0 μmol/L.

Operation table

1. Enzymatic reaction (Reagent 1 application solution is preheated in advance at 37℃)

	Non-enzyme tube	Enzyme tube
1 mmol/L GSH (μL)	40	40
Sample (μL)		40
Pre-heat the tubes at 37℃ water bath for 5 min. Preheat Reagent 1 application solution at 37℃ for 5 min at the same time.
Reagent 1 application solution (μL)	20	20
React at 37℃ water bath for 5 min accurately.
Reagent 2 (μL)	400	400
Sample (μL)	40

Mix fully and centrifuge at 3100 g for 10 min, then take 100 μL of supernatant for chromogenic reaction.

2. Chromogenic reaction

	Standard well	Non-enzyme well	Enzyme well
GSH standard solution with different concentrations (μL)	100
Supernatant (μL)		100	100
Reagent 3 (μL)	100	100	100
Reagent 4 (μL)	50	50	50

Mix fully and stand for 15 min at room temperature. Measure the OD values of each well at 412 nm with microplate reader.

Performance characteristics

Technical parameter

Detection range	34.34-1036.64 U	Average inter-assay CV	8.699999999999999%
Sensitivity	34.34 U	Average intra-assay CV	2.4%
Average recovery rate	104%

Standard curve

Dilute 100 μmol/L GSH standard solution with GSH standard application solution to a serial concentration. The recommended dilution gradient is as follows: 100, 80, 60, 50, 40, 20, 10, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using OD value of standard and correspondent concentration as y-axis and x-axis respectively. Create the standard curve with graph software (or EXCEL). The concentration of the sample can be calculated according to the formula based on the OD value of sample. The standard curve is: y= ax + b.

Example analysis

For Rat liver tissue, dilute the 10% rat liver tissue homogenate with PBS (0.01 M, pH 7.4) for 20 times, carry the assay according to the operation table.

The results are as follows:

standard curve: y=0.0038x-0.0013, the average OD value of Non-enzyme tube is 0.325, the average OD value of Enzyme tube is 0.213, the concentration of protein in sample is 13.99 mgprot/mL, and the calculation result is:

GSH-PX activity (U/mgprot)=(0.325-0.213+0.0013)÷0.0038×5÷5÷0.04÷13.99×20=1065.61U/mgprot

Detect mouse serum (dilute for 4 times), 10% rat liver tissue homogenate (the concentration of protein is 13.99 mgprot/mL dilute for 20 times), 10% aureum tissue homogenate (the concentration of protein is 1.59 mgprot/mL) and HepG2 cells (the concentration of protein is 5.02 mgprot/mL) according to the protocol, the result is as follows:

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