Solarbio 피루베이트 카르복실라제 (PC) 활동 분석 키트

피루브산 카르복실라제 (PC) 활동 분석 키트

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

운영장비: 분광 광도계

고양이 번호: BC0730

크기:50T/48S

구성요소:

용액 추출: 액체 110 밀리리터×1. 4℃에서 보관.

시약 I: 액체 30 밀리리터×1. 4℃에서 보관.

시약 II: 액체 10 밀리리터×1. 4℃에서 보관.

시약 III: 가루×1. -20℃에서 보관. Dissolve with 5 증류수 1mL, store at -20℃ after prepared.

시약 IV: 가루×1. -20℃에서 보관. Dissolve with 5 증류수 1mL, store at -20℃ after prepared.

시약 V: 액체 5 밀리리터×1. 4℃에서 보관.

시약 VI: 액체 15 μL×1. 4℃에서 보관.

Reagent VI Diluent Solution: 액체 10 밀리리터×1. 4℃에서 보관.

제품 설명:

Pyruvate carboxylase (PC, EC 6.4.1.1) is widely present in mitochondria of animals, molds and yeast, but is not found in plants and most bacteria. PC is the main postreaction for oxaloacetate, and is the first-rate- limiting enzyme in the gluconeogenesis process.

PC irreversibly catalyzes pyruvate, ATP, CO2 and water to oxaloacetate, ADP and Pi, malic dehydrogenase further catalyzes the formation of malic acid and NAD+ from acetoacetic acid and NADH. The enzyme activity of PC can be reflected by detecting the oxidation rate of NADH at 340 nm.

필요하지만 제공되지 않는 시약 및 장비:

Ultraviolet spectrophotometer, 욕조, 책상 원심분리기, 욕조, 조절 가능한 피펫, 1 mL 석영 큐벳, 모르타르/균질화기, 얼음과 증류수.

절차:

나. Complex extraction:

• Collecting 0.1 g of tissue or 5 백만개의 세포, 추가하다 1 mL의 추출 용액, grinding on ice with mortar/homogenizer.
• 원심분리기 1000 ×g 10 minutes at4℃,
• Take the supernatant to other tube and centrifuge at 11000 ×g 15 minutes at4℃.
• The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
• 추가하다 1 mL of Extract solution to the sediment, splitting with ultrasonic (힘 20%, work time 5s, 간격 10초, 반복하다 12 타임스), used to detect the enzyme activity of PC and protein content.

II. 결정 절차:

• Preheat ultraviolet spectrophotometer for 30 분, 파장을 조정하여 340 nm, 증류수로 0을 설정하다.
• Preheat Reagent I at 37℃ for 15 분.
• Diluent Reagent VI according to the volume ratio of Reagent VI: Reagent VI Diluent Solution = 1.6:660(V:V), prepare the reagent when it will be used.
• 작업 솔루션: make the solution as the volume ratio of Reagent II: 시약 III: Reagent IV= 2:1:1, prepare the reagent when it will be used.
• Add the following reagents in 1 mL 석영 큐벳:

III. 계산:

단위 정의: One unit of enzyme activity is defined as the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every milligram of protein.

PC Activity(U/mg 프로트)=[ΔA×Vrv÷(ε×d)×109]¶(VS×심폐소생술)÷T =1607×ΔA÷Cpr

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

로프: 총 반응량,1×10-3L; 대: 샘플량 (밀리리터), 0.05 밀리리터;

심폐소생술: 샘플 단백질 농도 (mg/mL); 티: 반응 시간 (분), 2 분;

109: 1 mol=109 nmol.

메모:

1. Take one or two different samples for prediction before test. It is recommended to dilute the crude enzyme solution with the Extract solution before the determination if the ΔA>0.8(if measuring with 96 well flat-bottom UV plate, ΔA>0.5). 하는 동안, extending the response time (5 minutes or 10 분) if ΔA <0.01.

2. The blank tube is a detection hole for detecting the quality of each reagent component, and normally that the change of ΔAB does not exceed 0.05.
3. The protein concentration of the sample needs to be determined by yourself. Since the Extract solution contains a relatively high protein concentration (~에 대한 1 mg/mL), the protein concentration of the Extract solution must be deducted when measuring the protein concentration of the sample.

4. It is recommended to use the sample protein concentration to calculate the enzyme activity. If the sample fresh weight is used to calculate, the enzyme activity of cytoplasmic extract needs to be measured, and the sum of supernatant and precipitation enzyme activity is the total enzyme activity.
5. Reagents in this kit are sufficient to complete 50 tube reactions.
6. Appendix: calculation formula of sample weight: (sample test number is50T/24S)

1) 상청액:

단위 정의: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.

PC Activity (U/g 중량) =[ΔA1×Vrv÷(ε×d)×109]¶(W²Ve×Vs)÷T =1607×ΔA1÷W

ΔA1: Supernatant absorbance;

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

로프: 총 반응량,1×10-3L; 대: 샘플량 (밀리리터), 0.05 밀리리터; 베: Extraction solution, 1 밀리리터;

심폐소생술: 샘플 단백질 농도 (mg/mL); 티: 반응 시간 (분), 2 분;

109: 1 mol=109 nmol;

여: 샘플 중량, g.

2) Sediment:

단위 정의: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.

PC Activity (U/g 중량) =[ΔA2×Vrv÷(ε×d)×109]¶(W²Ve×Vs)÷T =1607×ΔA2÷W

ΔA2: Sediment absorbance;

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

로프: 총 반응량,1×10-3L; 대: 샘플량 (밀리리터), 0.05 밀리리터;

베: Sediment heavy suspension volume, 1 밀리리터; 심폐소생술: 샘플 단백질 농도 (mg/mL); 티: 반응 시간 (분), 2 분;

109: 1 mol=109 nmol;

여: 샘플 중량, g.

3) Total 활동

Total activity is the sum of PC activity in supernatant and sediment. PC(U/g 중량)=1607×ΔA1÷W+1607×ΔA2÷W.

실험예:

1. 1 mL of Extract solution is added to 0.1 g of rabbit heart tissue for homogenization. The supernatant is diluted 100 times with Extract solution, and the precipitation was diluted 4 타임스. 그 다음에, measured by microquartzplate according to the determination steps, 상청액: the ΔAT = A1T – A2T = 1.104-0.856 =0.248, ΔAB = A1B – A2B = 1.021-0.988=0.033, Δ A1 = ΔAT – ΔAB = 0.248-0.033=0.215, precipitate: ΔAT = A1T – A2T = 1.07-0.716 =0.354, ΔAB= A1B- A2B = 1.021-0.988=0.033, ΔA2 = ΔAT- ΔAB =0.354-0.033= 0.321

상청액: the activity of PC (U/g 질량) = 1607 × Δ A1 ÷ W × 100 (희석비율) = 1607×0.215÷0.1× 100 = 345505 U/g 질량;

Precipitation: the enzyme activity of PC (U/g 질량) = 1607×ΔA2 ÷ W×4 (희석비율) = 1607×0.321÷ 01.

× 4=20633.88 U/g mass;

The total enzyme activity of PC (U/g 질량) = 1607×ΔA1÷W×100 (dilution) + 1607×ΔA2 ÷ W

=1607 × 0.215 ÷0.1×100+1607×0.321÷0.1×4=366138.88 U/g mass.

참고자료

[1] Esmail S. Kakey,Amez A. Ismael. Evaluation of Oxidative Stress Status in Aged Human in relation to some Diseases. International Conference on Pure and Applied Sciences. August 2018;

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