Solarbio Glutathionperoxidase (GSH-PX/GPX) Aktivitätstest-Kit

Notiz: Take two or three different samples for prediction before the test.

Betriebsausrüstung: Spektrophotometer

Katze nein: BC1190

Größe: 50T/24s

Komponenten:

Produktbeschreibung:

• Glutathione peroxidase (GPX), also known as GSH-Px or GPX, is a crucial peroxidase enzyme found widely in the body.
• GPX plays a significant role in catalyzing the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG) and in reducing toxic hydrogen peroxide to non-toxic hydroxyl compounds.
• The enzymatic action of GPX involves the oxidation of GSH by hydrogen peroxide, resulting in the production of GSSG.
• GSH reacts with DTNB to form compounds with characteristic absorption peaks at 412 nm.
• The decrease in absorbance at 412 nm serves as an indicator of GPX activity.

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung:

Spektrophotometer, balance, Tabelle Zentrifuge, 1 ML Glass Cuvette, Mörtel/Homogenisator, EP tube.

Verfahren

ICH. Probenvorbereitung:

Gewebe:

Accordance ratio Tissue weight (G): Lösung extrahieren (ml)=1:5~ 10 (Suggested 0.05g of tissue with 1mL of Extract solution), homogenate on ice bath. Zentrifuge bei 5000 rpm at 4℃ for 10 Protokoll, Nimm den Überstand, and place it on ice for test (If the supernatant is not clear, Zentrifuge für 3 Protokoll).

Bakterien oder Zellen

Amount of cells (104): Lösung extrahieren (ml): 500~1000:1 (Add 1mL of Extract solution to 5 Millionen Zellen), ultrasonic with ice bath to break cells(300W,3S, interval 7s,total time 3 Protokoll). Centrifuged at 5000 rpm at 4℃ for 10 Protokoll, take the supernatant and place it on ice for test (If the supernatant is not clear, Zentrifuge für 3 Protokoll).

Serumprobe: Detect directly

II. Bestimmung Verfahren:

1. Das Spektrophotometer vorheizen 30 Protokoll, Passen Sie die Wellenlänge an 412 nm, and set zero with distilled water.
2. The standard solution of 20 μmol/mL was diluted to 0.25 μmol/mL with the extraction solution. The standard solution of 100 μL is mixed with 400 μl Reagenz IV, and the concentration of the standard solution was 0.05 μmol/ml. The standard solution is prepared when the solution mixture is used.
3. Mix the 150 μL of sample with the 150 μLof reagent I and place it at room temperature for 5 Protokoll.
4. Operation table: (1.5 mL centrifugal tube with the following reagents in turn).

Zentrifuge bei 4000 rpm at room temperature for 5 minutes and take the supernatant into an EP tube.

Well mix. Then placed at room temperature for 15 Protokoll, die Absorption bei 412 nm is measured. The absorbance is recorded as AT, AC, ALS, and AB, respectively. Calculate ΔAT = AC – BEI, ΔAS= AS – AB.

III. Berechnung:

Calculation of inhibition percentage Inhibitory percentage =(AC-AT)/(AC-AB)×100%

As far as possible, the inhibition percentage of the sample is within the range of 30-70%, and the closer it is to 50%, the more accurate it is. If the inhibition percentage is less than 30% oder mehr als 70%, it is usually necessary to adjust the dosage and re-determine it. If the inhibition percentage is high, Die Probe sollte ordnungsgemäß verdünnt werden. If the inhibition percentage is low, the sample with a high concentration should be prepared again.

Calculation of GPX activity

• Proteinkonzentration:

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1nmol of GSH per minute in the reaction system, Jedes Milligramm Protein.

GPX (U/mg-Schutz) =ΔAT÷(ΔAS÷CS)×1000×VEV÷(Cpr×VSV)÷T=200×ΔAT÷ΔAS÷Cpr

• Probengewicht

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of GSH per minute in the reaction system, every gram of sample.

GPX (U/g-Gewicht) =ΔAT÷(ΔAS÷CS)×1000×VEV÷(VSV÷VTV×W)÷T=200×ΔAT÷ΔAS÷W

• Zellmenge

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1nmol of GSH per minute in the reaction system, every 104 Zellen.

GPX(U/104cell) =ΔAT÷(ΔAS÷CS)×1000×VEV÷(N×VSV÷VTV)÷T=200×ΔAT÷ΔAS÷N

• Liquid volume:

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of GSH per minute in the reaction system, every milliliter of liquid.

GPX (U/ml)=ΔAT÷(ΔAS÷CS)×1000×VEV÷VS÷T=200×ΔAT÷ΔAS.

CS: Concentration of standard mixtures, 0.08 μmol/ml;

VEV: Volume of the enzymatic reaction system, 1.25ml;

VSV: Sample volume contained in sample mixtures, 0.1 ml; VTV: Extraction solution volume, 1 ml;

Cpr: Überstandsproteinkonzentration, mg/ml;

T: Reaktionszeit, 5 Protokoll;

N: The amount of cells, tens of thousands; W: Probengewicht, G;

1000: 1 μmol=1000 nmol.

Notiz:

1. When the absorbance is greater than 1.2, it is suggested that the sample be determined after diluted with the extraction
2. It is recommended not to take too many samples at a time, to avoid the influence of too long testing time on color development, which may let the determination is not

Experimental instances：

• Take 0.1g of mouse liver, add 1mL of extract solution, homogenate, and grind. Take the supernatant, dilute it by 40 mal, and test according to the measurements Calculate AT=0.108, AC=0.303, AS=0.491AB=0.033,∆AT=AC-AT=0.195 ∆AS= AS-AB=0.458, calculate the enzyme activity according to sample weight:

GPX (U/g-Gewicht)=200×ΔAT÷ΔAS÷W×40（dilution ratio）=34061 U/g.

• Take 1g of poplar leaf, add 1mL of extract solution, homogenate, and grind. Calculate AT=0.220, AC=0.318, AS=0.491, AB=0.033, ∆AT=AC-AT=0.098,∆AS=AB-AB=0.458, calculate the enzyme activity according to sample weight:

GPX (U/g-Gewicht)=200×ΔAT÷ΔAS÷W=428 U/g.