Piruvato carboxilasa (ordenador personal) Kit de ensayo de actividad
Nota: Tome dos o tres muestras diferentes para predecir antes de la prueba..
Equipo de operación: espectrofotómetro
No gato: BC0730
Tamaño:50T/48S
Componentes:
Extraer solución: Líquido 110 mL×1. Almacenamiento a 4 ℃.
reactivo yo: Líquido 30 mL×1. Almacenamiento a 4 ℃.
Reactivo II: Líquido 10 mL×1. Almacenamiento a 4 ℃.
Reactivo III: Polvo × 1. Almacenamiento a -20 ℃. disolver con 5 ml de agua destilada, store at -20℃ after prepared.
Reactivo IV: Polvo × 1. Almacenamiento a -20 ℃. disolver con 5 ml de agua destilada, store at -20℃ after prepared.
Reactivo V: Líquido 5 mL×1. Almacenamiento a 4 ℃.
Reactivo VI: Líquido 15 µL×1. Almacenamiento a 4 ℃.
Reagent VI Diluent Solution: Líquido 10 mL×1. Almacenamiento a 4 ℃.
Descripción del Producto:
Pyruvate carboxylase (ordenador personal, CE 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 Nuevo Méjico.
Reactivos y equipos necesarios pero no suministrados:
Ultraviolet spectrophotometer, baño de agua, centrífuga de escritorio, baño de agua, pipeta ajustable, 1 mL quartz cuvette, mortero/homogeneizador, hielo y agua destilada.
Procedimiento:
I. Complex extraction:
- Collecting 0.1 g of tissue or 5 millones de células, agregar 1 mL de solución de extracto, grinding on ice with mortar/homogenizer.
- Centrifugar en 1000 ×g para 10 minutes at4℃,
- Take the supernatant to other tube and centrifuge at 11000 ×g para 15 minutes at4℃.
- The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
- Agregar 1 mL of Extract solution to the sediment, splitting with ultrasonic (fuerza 20%, tiempo de trabajo 5s, intervalo 10s, repetir 12 veces), used to detect the enzyme activity of PC and protein content.
II. Determinación procedimiento:
- Preheat ultraviolet spectrophotometer for 30 minutos, ajustar la longitud de onda a 340 Nuevo Méjico, poner a cero con agua destilada.
- Preheat Reagent I at 37℃ for 15 minutos.
- 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.
- Solución de trabajo: make the solution as the volume ratio of Reagent II: Reactivo III: Reagent IV= 2:1:1, prepare the reagent when it will be used.
- Add the following reagents in 1 mL quartz cuvette:
| Reactivo (µL) | tubo en blanco (B) | Tubo de ensayo (t) |
| reactivo yo | 450 | 450 |
| Solución de trabajo | 320 | 320 |
| Reactivo V | 80 | 80 |
| Reactivo VI | 100 | 100 |
| Muestra | – | 50 |
| Agua destilada | 50 | – |
| Add the above reagents to the 1 mL quartz cuvette in order, timing after add working solution, mezclar bien. Detect the absorbance at 340 nm at the time of 10 segundos, record as AT1 or AB1. Then place dishes with the reaction solution in a 37℃ water bath for 2 minutos. Take it out and wipe it clean, immediately measure the absorbance at the time of 130 segundos, which record as AT2 or AB2. ΔAT= AT1- AT2, ΔAB= AB1- AB2, ΔA= ΔAT-ΔAB. The blank tube only need to test once or twice. | ||
III. Cálculo:
Definición de unidad: 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(Prot. U/mg)=[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =1607×ΔA÷Cpr
mi: NADH molar extinction coefficient, 6.22×103 L/mol/cm; d: Light path of cuvette, 1 cm;
Soga: Volumen total de reacción,1×10-3 L; contra: Volumen de la muestra (mililitros), 0.05 mililitros;
RCP: Concentración de proteína de muestra (mg/mL); t: Tiempo de reacción (mín.), 2 minutos;
109: 1 mol=109 nmol.
Nota:
- 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). While, extending the response time (5 minutes or 10 minutos) if ΔA <0.01.
- 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.
- The protein concentration of the sample needs to be determined by yourself. Since the Extract solution contains a relatively high protein concentration (acerca de 1 mg/mL), the protein concentration of the Extract solution must be deducted when measuring the protein concentration of the sample.
- Se recomienda utilizar la concentración de proteína de la muestra para calcular la actividad enzimática.. Si se utiliza el peso fresco de la muestra para calcular, Es necesario medir la actividad enzimática del extracto citoplasmático., y la suma de la actividad enzimática del sobrenadante y la precipitación es la actividad enzimática total.
- Reagents in this kit are sufficient to complete 50 reacciones del tubo.
- Appendix: calculation formula of sample weight: (sample test number is50T/24S)
1) Flotante:
Definición de unidad: 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 (Peso U/g) =[ΔA1×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA1÷W
ΔA1: Supernatant absorbance;
mi: NADH molar extinction coefficient, 6.22×103 L/mol/cm; d: Light path of cuvette, 1 cm;
Soga: Volumen total de reacción,1×10-3 L; contra: Volumen de la muestra (mililitros), 0.05 mililitros; ve: Extraction solution, 1 mililitros;
RCP: Concentración de proteína de muestra (mg/mL); t: Tiempo de reacción (mín.), 2 minutos;
109: 1 mol=109 nmol;
W.: Peso de la muestra, gramo.
2) Sedimento:
Definición de unidad: 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 (Peso U/g) =[ΔA2×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA2÷W
ΔA2: Sediment absorbance;
mi: NADH molar extinction coefficient, 6.22×103 L/mol/cm; d: Light path of cuvette, 1 cm;
Soga: Volumen total de reacción,1×10-3 L; contra: Volumen de la muestra (mililitros), 0.05 mililitros;
ve: Sediment heavy suspension volume, 1 mililitros; RCP: Concentración de proteína de muestra (mg/mL); t: Tiempo de reacción (mín.), 2 minutos;
109: 1 mol=109 nmol;
W.: Peso de la muestra, gramo.
3) Total activity
Total activity is the sum of PC activity in supernatant and sediment. ordenador personal(Peso U/g)=1607×ΔA1÷W+1607×ΔA2÷W.
Ejemplo experimental:
- 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 veces. Entonces, measured by microquartzplate according to the determination steps, Flotante: 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
Flotante: the activity of PC (masa U/g) = 1607 × Δ A1 ÷ W × 100 (relación de dilución) = 1607×0.215÷0.1× 100 = 345505 masa U/g;
Precipitation: the enzyme activity of PC (masa U/g) = 1607×ΔA2 ÷ W×4 (relación de dilución) = 1607×0.321÷ 01.
× 4=20633.88 U/g mass;
The total enzyme activity of PC (masa U/g) = 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.
Referencias
[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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