Solarbio Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Aktivitätstest-Kit

Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Aktivitätstest-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Erkennungsinstrument: Spektrophotometer

Katze nein: BC2210

Größe: 25T/24 ; 50T/48S

Komponenten:

Lösung extrahieren: 60 ml×1. Speichern Sie bei 4 ℃.

Reagenz I: Pulver×1. Bei -20℃ lagern.

Reagenz II: 50 ml×1. Speichern Sie bei 4 ℃.

Reagenz III: 30 μL×1. Speichern Sie bei 4 ℃. The liquid is placed in the EP tube in the reagent bottle. According to the dosage and the volume ratio of Reagent III: distilled water of 3:100, gut mischen, use and prepare now.

Produktbeschreibung:

GAPDH (EC 1.2.1.12) catalyzes the oxidation of glyceraldehyde 3-phosphate to 1,3-diphosphoglyceride. It is the key enzyme of glycolysis pathway. It is closely related to the pathway of gluconeogenesis, the maintenance of blood glucose concentration and the occurrence of diabetes. It plays an important role in the disorders of glucose, lipid and protein metabolism.

3-phosphoglycerate kinase can catalyze the production of 1,3-diphosphoglyceride from triphosphate and ATP. GAPDH reversely catalyzes the formation of glyceraldehyde-3-phosphate, inorganic phosphorus and NAD+ from 1,3-diphosphoglyceride and NADH. The decrease of NADH measured at 340 nm can reflect the activity of GAPDH.

Required material

Desk centrifuge, ultraviolet spectrophotometer, Wasserbad konstanter Temperatur, mortar/ homogenizer, 1 ml-Quarzküvette, transferpettor, Eis und destilliertes Wasser.

Verfahren:

ICH. Probe Extraction:

1. Gewebeprobe:

According to the mass of the tissue (G): the volume of the Extract solution (ml) is 1: 5~ 10. Suggested 0.1 G Gewebe mit 1 ml Extraktlösung. Fully grind on ice, Zentrifuge bei 8000 g and 4℃ for 20 Mindest. Supernatant is placed on ice for test.

2. Bakterien oder Zellen:

According to the number of cells (104): the volume of the Extract solution (ml) is 500 ~ 1000: 1. Recommend 5 million with 1 mL of Extract Solution. Use ultrasonication to split bacteria or cells (Leistung 20% or 200W, Arbeitszeit 3s, Intervall 10s, Wiederholen Sie 30 mal). Zentrifuge bei, 8000 g and 4℃ for 10 Mindest. Supernatant is placed on ice for test.

3. Serum (Plasma): direct measurement.

II. Bestimmung Verfahren:

• Preheat the ultraviolet spectrophotometer 30 Mindest, adjust wavelength to340 nm, Mit destilliertem Wasser auf Null stellen.
• Preparation of working solution: pour all Reagent II into one bottle of Reagent I. Fully dissolved. Preheat a certain amount of 37℃ (Säugetier) oder 25 ℃ (andere Arten) für 10 min as required. The unused reagents shall be stored at — 20℃ after sub charging. Avoid repeated freezing and thawing.
• Fügen Sie Reagenzien mit der folgenden Liste hinzu:

III. Berechnung:

Calculated by micro quartz cuvette

• Calculated by protein concentration:

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every mg protein.

GAPDH activity (U/mg-Schutz) =ΔA÷(ε×d)×VRV×109÷(VS×Cpr)÷T=1072×ΔA÷Cpr

• Calculated by sample weight

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every g sample.

GAPDH activity (U/g fresh weight) =ΔA÷(ε×d)×VRV×109÷(VS×W÷VST)÷T= 1072×ΔA÷W

• Calculated by bacteria or cell amount:

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every 104 bacteria or cells.

GAPDH activity (U/104 -Zelle) =ΔA÷(ε×d)×VRV×109÷(VS×500÷VST)÷T = 2.14×ΔA

• Calculated by volume of culture medium:

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every mL liquid.

GAPDH activity (U/ml) =ΔA÷(ε×d)×VRV×109÷VS÷T= 1072×ΔA

e: molar extinction coefficient of NADH: 6.22×103 L/mol/cm; D: light path of cuvette, 1 cm;

Kabel: Gesamtreaktionsvolumen, 0.001 L;

Vs: sample volume in reaction system, 0.03 ml; VST: volume of extraction solution added, 1 ml; Cpr: Probenproteinkonzentration, mg/ml;

W, Probenmasse, G;

T: Reaktionszeit, 5 Mindest;

109: conversion factor, 1 mol = 109 Nmol;

500: Number of cells, 5 Million.

Notiz:

1. When A1 is less than 0.8 or ΔA is greater than 0.7, it is recommended to dilute the sample before determination.
2. The blank tube is a test tube for testing the quality of each reagent component. Under normal conditions, the change does not exceed01.

Experimentelles Beispiel:

1. Take 0.1g of rabbit kidney, hinzufügen 1 ml Extraktlösung, homogenize in ice bath, then centrifuge at 8000g, 4℃ für 20 Mindest, take the supernatant and put it on ice, then operate with micro quartz cuvette according to the determination steps, measure and calculate: ΔAT=A1T-A2T =0.815-0.158=0.657, ΔAB= A1B – A2B = 0.865-0.864=0.001, ΔA = ΔAT – ΔAB = 656.

GAPDH activity (U/g-Masse) = 1072×ΔA ÷ W = 7032.32 U/g-Masse.

2. Take 0.1g of clover, hinzufügen 1 ml Extraktlösung, homogenize it in ice bath, then centrifuge it in 8000g and 4℃ for 20 Mindest, take the supernatant and put it on ice, then operate according to the determination steps, measure and calculate it with micro quartz cuvette, ΔAT = A1T – A2T =1.026-1.017=0.009, ΔAB= A1B – A2B =0.865-0.864=0.001, ΔA = ΔAT – ΔAB=008.

GAPDH activity (U/g-Masse) = 1072 × ΔA ÷ W =85.76 U/g mass.

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