Plant Dehydrogenase (Pdhi) Kit de ensayo de actividad
Nota: Tome dos o tres muestras diferentes para predecir antes de la prueba..
Equipo de operación: Microplate Reader/ Spectrophotometer
No gato: BC3125
Tamaño:100T/48S
Componentes:
reactivo yo: Polvo×2. Almacenamiento a 4 ℃. Dissolve one bottle of powder in water to make 50 mL before use, prepare the solution when it will be use. Store at 4℃ and protect from light after prepared.
Reactivo II: Líquido 100 mL×2. Almacenamiento a 4 ℃.
Reactivo III: Ethyl acetate, required but not provided.
Descripción del Producto:
The activity of plant dehydrogenase (Pdhi) is largely reflecting the active state of the organism, which can directly indicate the ability of biological cells to degrade its matrix.
The hydrogen acceptor 2,3,5-triphenyl tetrazolium chloride (TTC) generates red triphenylformazone (TFF) after receiving hydrogen during cell respiration. TFF has a characteristic absorption peak at 485 Nuevo Méjico, the PDHA activity can quantified by measuring the absorbance at 485 Nuevo Méjico.
Reactivos y equipos necesarios pero no suministrados:
Microplate Reader or spectrophotometer, baño de agua, centrífuga de escritorio, baño de agua, pipette, cubeta de microvidrio/placa de fondo plano de 96 pocillos (non-polystyrene/polypropylene material), mortero/homogeneizador, ethyl acetate (express delivery is not allowed), hielo y agua destilada.
Procedimiento:
I. Complex extracción:
Recolectar 0.1 g de tejido, lavar 3 o 4 times with double steam water, blot dry with filter paper and set aside.
II. Determinación procedimiento:
- Preheat spectrophotometer/ microplate reader for 30 minutos, ajustar la longitud de onda a 485 Nuevo Méjico, set zero with ethyl acetate.
- Add the following reagents in 5 mL EP tubes:
| Reactivo | Contrast tube (C.A) | Tubo de ensayo (EN) |
| Muestra (gramo) | 0.1 | 0.1 |
| reactivo yo (mililitros) | – | 1 |
| Reactivo II (mililitros) | 2 | 1 |
| Mix thoroughly and stand in dark for 3 hours at 37℃, ice bath for 5 minutes immediately after take out. Discard the filtrate, blot dry the sample with filter paper, place in mortar / homogenizer. | ||
| Reactivo III (mililitros) | 1 | 1 |
III. Cálculo:
A. micro glass cuvette (light path of the cuvette, 1 cm)
Definición de unidad: One unit of enzyme activity is defined as the amount of enzyme increases the absorbance of every 0.01 for per hour every mg tissue protein in the reaction system.
PDHA Activity(U/g/h) =ΔA÷0.01÷T÷W=33×ΔA÷W
B. 96 buen plato (light path of the cuvette, 0.6 cm)
Definición de unidad: One unit of enzyme activity is defined as the amount of 1 mg of tissue increases the absorbance of every 0.005 for per hour in the reaction system.
PDHA Activity(U/g/h) =ΔA÷0.005÷T÷W=66.7×ΔA÷W
t: Tiempo de reacción (h), 3 horas; W.: Peso de la muestra, gramo.
Nota
- After prepared, Reagent I should store at 4℃, protect from light and used within one week. If it turns red, it cannot be used.
- Reagent III is volatile and toxic. For your health, please wear lab clothes, masks, and latex gloves.
- Ice bath to stop the reaction immediately after the dark reaction was completed. Discard the filtrate, blot dry the sample with filter paper as much as possible.
- Tome dos o tres muestras diferentes para predecir antes de la prueba.. Dilute the supernatant if the absorbance is higher, multiplicar los tiempos diluidos en la fórmula.
- If test with a 96 well flat-bottom plate, polystyrene, or polypropylene material 96 well flat-bottom plate is not recommended.
Ejemplo experimental:
- Weigh 1g aloe leaves, wash them with double distilled water for 3–4 times, absorb the water with filter paper, operate according to the determination steps, measure and calculate with 96 buen plato, ΔA=AT-AC= 0.244-0.146 = 0.098, calculate the enzyme activity.
Dehydrogenase activity (masa U/g) = 66.7×ΔA÷W = 6.5366 masa U/g.
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