Deidrogenasi vegetale (PDHA) Kit di analisi dell'attività
Nota: Prendi due o tre campioni diversi per la previsione prima del test.
Attrezzatura operativa: Microplate Reader/ Spectrophotometer
Gatto n: BC3125
Misurare:100T/48S
Componenti:
Reagente I: Polvere×2. Conservazione 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.
Reagente II: Liquido 100 mL×2. Conservazione a 4 ℃.
Reagente III: Ethyl acetate, required but not provided.
Descrizione del prodotto:
The activity of plant dehydrogenase (PDHA) 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 nm, the PDHA activity can quantified by measuring the absorbance at 485 nm.
Reagenti e attrezzature necessari ma non forniti:
Microplate Reader or spectrophotometer, bagnomaria, centrifuga da tavolo, bagnomaria, pipette, microcuvetta in vetro/piastra a fondo piatto da 96 pozzetti (non-polystyrene/polypropylene material), mortaio/omogeneizzatore, ethyl acetate (express delivery is not allowed), ghiaccio e acqua distillata.
Procedura:
IO. Complex estrazione:
Raccogliere 0.1 g di tessuto, lavare 3 O 4 times with double steam water, blot dry with filter paper and set aside.
II. Determinazione procedura:
- Preheat spectrophotometer/ microplate reader for 30 minuti, regolare la lunghezza d'onda su 485 nm, set zero with ethyl acetate.
- Aggiungere i seguenti reagenti 5 ML EP Tubi:
| Reagente | Tubo di contrasto (Ac) | Provetta (A) |
| Campione (G) | 0.1 | 0.1 |
| Reagente I (ml) | – | 1 |
| Reagente II (ml) | 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. | ||
| Reagente III (ml) | 1 | 1 |
III. Calcolo:
UN. micro glass cuvette (light path of the cuvette, 1 cm)
Definizione di unità: 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 bene piatto (light path of the cuvette, 0.6 cm)
Definizione di unità: 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: Tempo di reazione (H), 3 ore; W: Peso del campione, G.
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.
- Prendi due o tre campioni diversi per la previsione prima del test. Dilute the supernatant if the absorbance is higher, multiply dilute times in the formula.
- If test with a 96 piastra a fondo ben piatto, polystyrene, or polypropylene material 96 well flat-bottom plate is not recommended.
Esempio sperimentale:
- 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 bene piatto, ΔA=AT-AC= 0.244-0.146 = 0.098, calculate the enzyme activity.
Dehydrogenase activity (Massa U/g) = 66.7×ΔA÷W = 6.5366 Massa U/g.
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Recensioni
Non ci sono ancora recensioni.