Solarbio Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Activity Assay Kit

Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Activity Assay Kit

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

Detection instrument: Spectrophotometer

Cat No: BC2210

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

Components:

Extract solution: 60 mL×1. Store at 4℃.

Reagent I: Powder×1. Store at -20℃.

Reagent II: 50 mL×1. Store at 4℃.

Reagent III: 30 μL×1. Store at 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, mix well, use and prepare now.

Product Description:

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, constant temperature water bath, mortar/ homogenizer, 1 mL quartz cuvette, transferpettor, ice and distilled water.

Procedure:

I. Sample Extraction:

1. Tissue sample:

According to the mass of the tissue (g): the volume of the Extract solution (mL) is 1: 5~10. Suggested 0.1  g of tissue with 1 mL of Extract solution. Fully grind on ice, centrifuge at 8000 g and 4℃ for 20 min. Supernatant is placed on ice for test.

2. Bacteria or cells:

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 (power 20% or 200W, work time 3s, interval 10s, repeat for 30 times). Centrifuge at, 8000 g and 4℃ for 10 min. Supernatant is placed on ice for test.

3. Serum (plasma): direct measurement.

II. Determination procedure:

• Preheat the ultraviolet spectrophotometer 30 min, adjust wavelength to340 nm, set zero with distilled water.
• Preparation of working solution: pour all Reagent II into one bottle of Reagent I. Fully dissolved. Preheat a certain amount of 37℃ (mammal) or 25℃ (other species) for 10 min as required. The unused reagents shall be stored at — 20℃ after sub charging. Avoid repeated freezing and thawing.
• Add reagents with the following list:

III. Calculation:

    Calculated by micro quartz cuvette

• Calculated by protein concentration:

Unit definition: 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

Unit definition: 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:

Unit definition: 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 cell) =ΔA÷(ε×d)×VRV×109÷(VS×500÷VST)÷T = 2.14×ΔA

• Calculated by volume of culture medium:

Unit definition: 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

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

VRV: total reaction volume, 0.001 L;

VS: sample volume in reaction system, 0.03 mL; VST: volume of extraction solution added, 1 mL; Cpr: sample protein concentration, mg/mL;

W, sample mass, g;

T: reaction time, 5 min;

109: conversion factor, 1 mol = 109 nmol;

500: Number of cells, 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.

Experimental example:

1. Take 0.1g of rabbit kidney, add 1 mL of Extract solution, homogenize in ice bath, then centrifuge at 8000g, 4℃ for 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 mass) = 1072×ΔA ÷ W = 7032.32 U/g mass.

2. Take 0.1g of clover, add 1 mL of Extract solution, 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 mass) = 1072 × ΔA ÷ W =85.76 U/g mass.

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