ソーラーバイオ スーパーオキシド ジスムターゼ (SOD) 活性アッセイキット

Superoxide Dismutase (SOD) 活性アッセイキット

注記: 予測する必要がある 2-3 正式な決定前の大きな差異のサンプル.

操作装置: 分光光度計

カタログ番号: BC0170

サイズ: 50T/24S

コンポーネント:

抽出試薬: 60 mL×1. 4℃で保存.

試薬Ⅰ: 15 mL×1. 4℃で保存.

試薬Ⅱ: 160 μL×1. 4℃で保存. Mix by pipetting after centrifugation.

試薬Ⅲ: 11 mL×1. 4℃で保存.

試薬Ⅳ: パウダー×1. 4℃で保存.

試薬V: 2 mL×1. 4℃で保存. Add Reagent Ⅳ to Reagent V before use and shake with an oscillator to mix thoroughly. It can be stored 3 月.

製品説明:

Superoxide dismutase (SOD, EC 1.15.1.1) 動物に広く見られる, 植物, 微生物と培養細胞. It catalyzes the superoxide anion to form H2O2 and O2. SOD is not only the superoxide anion scavenging enzyme, but also the main H2O2 producing enzyme, which plays an important role in the biological antioxidant system.

Superoxide anion (O2-) is produced by the xanthine and xanthine oxidase reaction system. O2- can reduce blue tetrazole to create blue formazan, which has absorbance in 560 nm. SOD can remove O2- and inhibit the formation of methionine. The darker the blue color of the reaction solution, the lower the activity of SOD. The lighter the blue color of the reaction solution, the higher the activity of SOD.

必要だが提供されていない試薬と装置:

分光光度計, 卓上遠心分離機, 移送ピペット, 1 mLガラスキュベット, モルタル/ホモジナイザー, 氷と蒸留水.

Operation steps:

私. サンプルの準備:

1. 細菌とか細胞とか: collecting bacteria or cells into the centrifuge tube, discard supernatant after centrifugation. According to the proportion of bacteria or cells (104 細胞): 抽出液の量 (mL) の 1:5-10 to extract. It is suggested that 5 million of bacteria or cells amount with 1mL of Extraction reagent. Splitting the bacteria or cells with ultrasonication (氷の上に置かれた, ultrasonic power 200W or 20%, working time 3s, 間隔10秒, 繰り返します 30 回). で遠心分離します。 8000 ×gの場合 10 4℃で3分間不溶物を除去, 試験前に上清を氷上に置きます.
2. 組織: according to the proportion of tissue weight (g): 抽出液の量 (mL) の 1:5-10 to extract. It is suggested that 0.1 g of tissue with 1 mL of Extraction reagent and fully homogenized on ice.
3. で遠心分離します。, 8000 ×gの場合 10 4℃で3分間不溶物を除去, 試験前に上清を氷上に置きます.
4. 血清 (プラズマ) サンプル: detect sample directly.

Ⅱ. 決定 手順:

1. 分光光度計を予熱します。 30 分, 波長を調整して 560 nm を測定し、蒸留水でゼロに設定します.
2. Keep Reagent Ⅰ, 試薬Ⅲ, Reagent V in water bath for more than 5 37℃で3分(哺乳類) または

25℃ (他の種).

3. 次のリストを使用して試薬を追加します:

Mix thoroughly and the mixture is incubated at room temperature for 30 分. Add the mixture into 1mL glass cuvette, and detect the absorbance value of each tube at 560 nm. ΔAT=AT-AC，ΔAB=AB1-AB2. If there is precipitation at the bottom, mix thoroughly and then measure.

Ⅲ. 計算:

1. Inhibition percentage:

Inhibition percentage=[ΔAB -ΔAT]÷ΔAB× 100%

The inhibition percentage should be in 30%~70% (the value close to 50% will have a more accurate result). If the calculated inhibition percentage is less than 30% またはそれ以上 70%, it is usually necessary to adjust the sample addition amount and re determine. If the percentage of inhibition is too high, サンプルは適切に希釈する必要があります. If the percentage of inhibition is too low, the sample should be reprepared with a higher concentration.

2. 単位の定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the inhibition of 50% in the reaction system of the above xanthine
3. 計算

あ. 血清 (プラズマ)サンプル

SOD (U/mL)=[P÷(1-P)×Vrv]÷Vs×F=11.4×P÷(1-P)×F

B. 組織, 細菌または培養細胞

ある) タンパク質濃度:

SOD (U/mL prot) = [P÷(1-P)×Vrv]÷(VS×CPR)×F=11.4×P÷(1-P)÷Cpr×F

b) サンプル重量

SOD (U/g重量) = [P÷(1-P)×Vrv]÷(W×Vs÷Vsv)×F=11.4×P÷(1-P)÷W×F

c) Bacteria or cell amount

SOD (U/104セル)=[P÷(1-P)×Vrv]÷(500×Vs÷Vsv)×F=0.0228×P÷(1-P)×F

ロープ: 総反応量, 1.026 mL; 対: サンプル量, 0.09 mL;

VSv: 抽出量, 1 mL;

心肺蘇生法: サンプルタンパク質濃度, mg/mL; W: サンプル重量, g;

500: 細菌と細胞の総数, 5 百万. P: Inhibition percentage, %;

F: Sample dilution multiple.

注記:

1. The Sample and Reagent Ⅱ should be placed on ice when
2. When there are many samples, the working solution (including Reagent I, II and III) can be configured according to the table. Reagent V must be added
3. After the reaction completed, there may be precipitation formed, which can be determined after

実験例:

1. 1 g of Echinochloa crusgalli is added into 1 mL of Extraction reagent for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT = AT – AC = 0.335-0.012 = 0.323, ΔAB = AB1 – AB2 = 0.957-0.003 = 0.954. Inhibition percentage = (ΔAB- ΔAT)÷ ΔAB × 100% = 72%, and the enzyme activity is calculated according to the sample mass.

SOD activity (U/g質量) = 11.4 × Inhibition percentage (1-Inhibition percentage) × W = 293.14 U/g質量.

2. 1 mL of Extraction reagent is added to 0.1 g of rat spleen for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT= AT- AC = 0.563-0.213 = 0.35, ΔAB=AB1- AB2= 0.957-0.003 = 0.954, inhibition percentage = (ΔAB -ΔAT)÷ ΔAB×100% =31%

SOD activity (U/g質量) = 11.4 × Inhibition percentage (1-Inhibition percentage) ×W = 196.71 U/g質量.

3. 10 million cells is extracted and centrifuged by adding 1 抽出試薬 mL, and then the operation is performed according to the determination steps. The results are as follows: ΔAT =AT – AC=614-0.015 = 0.599, ΔAB=AB1- AB2= 0.944-0.005 = 0.939, inhibition percentage = (ΔAB- ΔAT) ×ΔAB× 100% = 36.21%

SOD activity (U/104セル) = Inhibition percentage ÷ (1-Inhibition percentage)× VTS]÷(1000× VS ÷ VTS) = 0.0065 U/104セル.

参考文献:

• スピッツ DR, オバリー L W. An assay for superoxide dismutase activity in mammalian tissue homogenates[J]. 分析生化学, 1989,179(1):8-18.
• マサヤス・マサヤス, Y ヒロシ. 臨床用スーパーオキシドジスムターゼ活性の簡易測定法[J]. クリニカ チミカ アクタ, 1979,92(3):337-342.

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