Solarbio Glycéraldéhyde-3-phosphate Déshydrogénase(GAPDH) Kit de test d'activité

Glycéraldéhyde-3-phosphate déshydrogénase(GAPDH) Kit de test d'activité

Note: Prenez deux ou trois échantillons différents pour la prédiction avant le test.

Instrument de détection: Spectrophotomètre

Chat non: BC2210

Taille: 25T/24 ; 50T/48S

Composants:

Extraire la solution: 60 mL×1. Conserver à 4℃.

Réactif I: Poudre × 1. Conserver à -20℃.

Réactif II: 50 mL×1. Conserver à 4℃.

Réactif III: 30 µL×1. Conserver à 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, bien mélanger, use and prepare now.

Description du produit:

GAPDH (CE 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, bain d'eau à température constante, mortar/ homogenizer, 1 CUVETTE ML, agent de transfert, glace et eau distillée.

Procédure:

je. Échantillon Extraction:

1. Échantillon de tissu:

According to the mass of the tissue (g): Le volume de la solution d'extrait (ml) est 1: 5~10. Suggested 0.1 g de tissu avec 1 mL de solution d'extrait. Fully grind on ice, centrifuger à 8000 g and 4℃ for 20 min. Supernatant is placed on ice for test.

2. Bactéries ou cellules:

According to the number of cells (104): Le volume de la solution d'extrait (ml) est 500 ~ 1000: 1. Recommend 5 million with 1 ml de solution d'extrait. Use ultrasonication to split bacteria or cells (pouvoir 20% ou 200W, temps de travail 3s, intervalle 10s, répéter pour 30 fois). Centrifuger à, 8000 g and 4℃ for 10 min. Supernatant is placed on ice for test.

3. Sérum (plasma): direct measurement.

II. Détermination procédure:

• Preheat the ultraviolet spectrophotometer 30 min, adjust wavelength to340 nm, mettre à zéro avec de l'eau distillée.
• Preparation of working solution: pour all Reagent II into one bottle of Reagent I. Fully dissolved. Preheat a certain amount of 37℃ (mammifère) ou 25 ℃ (autres espèces) pour 10 min as required. The unused reagents shall be stored at — 20℃ after sub charging. Évitez la congélation et la décongélation répétées.
• Ajoutez des réactifs avec la liste suivante:

Nom du réactif (µL)	Tube à essai (T)	Tube vierge (B)
Échantillon	30
Eau distillée		30
Réactif III	20	20
Solution de travail	950	950
Ajoutez les réactifs ci-dessus dans le 1 CUVETTE DE QUARTZ ML. Bien mélanger. Measure the absorbance value A1 at 340 nm for 10s. Quickly put it into a water bath or incubator at 37℃ (mammifère) ou 25 ℃ (autres espèces) pour 5 min. Take out and dry it quickly. Measure the absorbance value A2 for 5min10s. Calculate ΔAT= A1T- A2T, ΔAB= A1B- A2B, ΔA = ΔAT – ΔAB. Blank tube only needs to test 1–2 times.

III. Calcul:

Calculated by micro quartz cuvette

• Calculated by protein concentration:

Définition de l'unité: 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 prot) = ΔA ÷(ε × d)× VRV × 109 ÷(VS×Cpr)÷T=1072×ΔA÷Cpr

• Calculated by sample weight

Définition de l'unité: 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 (Poids frais U / g) = ΔA ÷(ε × d)× VRV × 109 ÷(VS×W÷VST)÷T= 1072×ΔA÷W

• Calculated by bacteria or cell amount:

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, chaque 104 bactéries ou cellules.

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

• Calculated by volume of culture medium:

Définition de l'unité: 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;

la corde: Volume de réaction total, 0.001 L;

CONTRE: sample volume in reaction system, 0.03 ml; Vst: volume of extraction solution added, 1 ml; Cpr: concentration en protéines de l'échantillon, mg/ml;

W, masse de l'échantillon, g;

T: temps de réaction, 5 min;

109: conversion factor, 1 mol = 109 nmol;

500: Nombre de cellules, 5 million.

Note:

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.

Exemple expérimental:

1. Take 0.1g of rabbit kidney, ajouter 1 mL de solution d'extrait, homogenize in ice bath, then centrifuge at 8000g, 4℃ pour 20 min, 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, ajouter 1 mL de solution d'extrait, homogenize it in ice bath, then centrifuge it in 8000g and 4℃ for 20 min, 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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