Solarbio 글리세르알데히드-3-인산염 탈수소효소(갭DH) 활동 분석 키트

글리세르알데히드-3-인산염 탈수소효소(갭DH) 활동 분석 키트

메모: 테스트 전에 예측을 위해 2~3개의 서로 다른 샘플을 채취합니다..

탐지 장비: 분광 광도계

고양이 번호: BC2210

크기: 25T/24 ; 50T/48S

구성요소:

용액 추출: 60 밀리리터×1. 4℃에서 보관.

시약 I: 가루×1. -20℃에 보관.

시약 II: 50 밀리리터×1. 4℃에서 보관.

시약 III: 30 μL×1. 4℃에서 보관. 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, 잘 섞다, use and prepare now.

제품 설명:

갭DH (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, 항온수욕, 모르타르/균질화기, 1 mL 석영 큐벳, 트랜스페터, 얼음과 증류수.

절차:

나. 견본 Extraction:

1. 조직 샘플:

According to the mass of the tissue (g): 추출 용액의 부피 (밀리리터) is 1: 5~10. Suggested 0.1 g의 티슈 1 mL의 추출 용액. Fully grind on ice, 원심분리기 8000 g and 4℃ for 20 분. Supernatant is placed on ice for test.

2. 박테리아 또는 세포:

According to the number of cells (104): 추출 용액의 부피 (밀리리터) is 500 ~ 1000: 1. Recommend 5 million with 1 mL의 추출 용액. Use ultrasonication to split bacteria or cells (힘 20% or 200W, 작업시간 3초, 간격 10초, 반복하다 30 타임스). 원심분리기, 8000 g and 4℃ for 10 분. Supernatant is placed on ice for test.

3. 혈청 (혈장): direct measurement.

II. 결정 절차:

• Preheat the ultraviolet spectrophotometer 30 분, adjust wavelength to340 nm, 증류수로 0을 설정하다.
• Preparation of working solution: pour all Reagent II into one bottle of Reagent I. Fully dissolved. Preheat a certain amount of 37℃ (포유 동물) 또는 25℃ (다른 종) ~을 위한 10 min as required. The unused reagents shall be stored at — 20℃ after sub charging. Avoid repeated freezing and thawing.
• 다음 목록으로 시약을 추가하세요.:

III. 계산:

Calculated by micro quartz cuvette

• Calculated by protein concentration:

단위 정의: 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 프로트) =ΔA π(ε×d)×VRV×109¶(VS×심폐소생술)÷T=1072×ΔA÷Cpr

• Calculated by sample weight

단위 정의: 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 신선 중량) =ΔA π(ε×d)×VRV×109¶(VS×W÷VST)÷T= 1072×ΔA÷W

• Calculated by bacteria or cell amount:

단위 정의: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, 모든 104 박테리아 또는 세포.

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

• Calculated by volume of culture medium:

단위 정의: 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 π(ε×d)×VRV×109÷VS÷T= 1072×ΔA

이자형: molar extinction coefficient of NADH: 6.22×103L/몰/cm; 디: light path of cuvette, 1 센티미터;

VRV: total reaction volume, 0.001 엘;

VS: sample volume in reaction system, 0.03 밀리리터; VST: volume of extraction solution added, 1 밀리리터; 심폐소생술: 샘플 단백질 농도, mg/mL;

여, sample mass, g;

티: 반응 시간, 5 분;

109: conversion factor, 1 mol = 109 nmol;

500: Number of cells, 5 백만.

메모:

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.

실험예:

1. Take 0.1g of rabbit kidney, 추가하다 1 mL의 추출 용액, homogenize in ice bath, then centrifuge at 8000g, 4℃ for 20 분, 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 질량) = 1072×ΔA ÷ W = 7032.32 U/g 질량.

2. Take 0.1g of clover, 추가하다 1 mL의 추출 용액, homogenize it in ice bath, then centrifuge it in 8000g and 4℃ for 20 분, 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 질량) = 1072 × ΔA ÷ W =85.76 U/g mass.

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