植物デヒドロゲナーゼ (PDHA) 活性アッセイキット
注記: テスト前に予測のために 2 つまたは 3 つの異なるサンプルを取得します.
操作装置: Microplate Reader/ Spectrophotometer
カタログ番号: BC3125
サイズ:100T/48S
コンポーネント:
試薬I: パウダー×2. 4℃で保存. 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.
試薬II: 液体 100 mL×2. 4℃で保存.
試薬Ⅲ: Ethyl acetate, required but not provided.
製品説明:
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.
必要だが提供されていない試薬と装置:
Microplate Reader or spectrophotometer, 水浴, 卓上遠心分離機, 水浴, pipette, マイクロガラスキュベット/96ウェル平底プレート (non-polystyrene/polypropylene material), モルタル/ホモジナイザー, ethyl acetate (express delivery is not allowed), 氷と蒸留水.
手順:
私. Complex 抽出:
集める 0.1 組織グラム, 洗う 3 または 4 times with double steam water, blot dry with filter paper and set aside.
Ⅱ. 決定 手順:
- Preheat spectrophotometer/ microplate reader for 30 分, 波長を調整して 485 nm, set zero with ethyl acetate.
- 次の試薬を追加します。 5 mL EP tubes:
| 試薬 | Contrast tube (Ac) | 試験管 (で) |
| サンプル (g) | 0.1 | 0.1 |
| 試薬I (mL) | – | 1 |
| 試薬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. | ||
| 試薬Ⅲ (mL) | 1 | 1 |
Ⅲ. 計算:
あ. micro glass cuvette (light path of the cuvette, 1 cm)
単位の定義: 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 ウェルプレート (light path of the cuvette, 0.6 cm)
単位の定義: 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: 反応時間 (h), 3 時間; W: サンプル重量, g.
注記
- 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.
- テスト前に予測のために 2 つまたは 3 つの異なるサンプルを取得します. Dilute the supernatant if the absorbance is higher, 式の希釈時間を掛けます.
- If test with a 96 井戸平底プレート, polystyrene, or polypropylene material 96 well flat-bottom plate is not recommended.
実験例:
- Weigh 1g aloe leaves, wash them with double distilled water for 3–4 times, absorb the water with filter paper, 決定手順に従って動作します, measure and calculate with 96 ウェルプレート, ΔA=AT-AC= 0.244-0.146 = 0.098, calculate the enzyme activity.
Dehydrogenase activity (U/g質量) = 66.7×ΔA÷W = 6.5366 U/g質量.
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BC2010/BC2015 Glycollic Oxidase Activity Assay Kit
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