Plant Dehydrogenase (PDHI) Kit Uji Aktivitas
Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.
Peralatan Operasi: Microplate Reader/ Spectrophotometer
Kucing No: BC3125
Ukuran:100T/48S
Komponen:
Reagen I: Bedak×2. Penyimpanan pada suhu 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.
Reagen II: Cairan 100 mL×2. Penyimpanan pada suhu 4℃.
Reagen III: Ethyl acetate, diperlukan tetapi tidak disediakan.
Deskripsi Produk:
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 nm, the PDHA activity can quantified by measuring the absorbance at 485 nm.
Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:
Microplate Reader or spectrophotometer, mandi air, mesin sentrifugal meja, mandi air, pipette, kuvet kaca mikro/pelat dasar datar sumur 96 (non-polystyrene/polypropylene material), mortir/homogenizer, ethyl acetate (express delivery is not allowed), es dan air suling.
Prosedur:
SAYA. Complex extraction:
Mengumpulkan 0.1 g tisu, wash 3 atau 4 times with double steam water, blot dry with filter paper and set aside.
II. Tekad prosedur:
- Preheat spectrophotometer/ microplate reader for 30 menit, sesuaikan panjang gelombangnya 485 nm, set zero with ethyl acetate.
- Add the following reagents in 5 mL EP tubes:
| Reagen | Contrast tube (Ac) | Tabung reaksi (PADA) |
| Sampel (G) | 0.1 | 0.1 |
| Reagen I (ml) | – | 1 |
| Reagen 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. | ||
| Reagen III (ml) | 1 | 1 |
AKU AKU AKU. Perhitungan:
A. micro glass cuvette (light path of the cuvette, 1 cm)
Definisi satuan: 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 piring sumur (light path of the cuvette, 0.6 cm)
Definisi satuan: 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: Waktu reaksi (H), 3 jam; W: Berat sampel, G.
Catatan
- 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.
- Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian. Dilute the supernatant if the absorbance is higher, multiply dilute times in the formula.
- If test with a 96 well flat-bottom plate, polystyrene, or polypropylene material 96 well flat-bottom plate is not recommended.
Contoh eksperimental:
- 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 piring sumur, 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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