Solarbio琥珀酸去氫酶 (SDH) 活性測定試劑盒

琥珀酸脫氫酶 (SDH) 活性測定試劑盒

筆記: 測試前取兩個或三個不同的樣本進行預測.

檢測設備: 分光光度計

目錄號: BC0950

尺寸: 50T/48S

成分

試劑一: 60 毫升×1, store at -20℃. 試劑二: 0.6 毫升×1, store at -20℃. 試劑三: 5 毫升×1, 4℃保存.

試劑四: 粉末×1, 4℃保存. When the solution will be used, add it into Reagent III to dissolve for use.

試劑V: 粉末×1, 4℃保存. 添加 4 mL of distilled water when the solution will be used, the unused reagents are stored at 4℃.

試劑六: 粉末×1, store at -20℃. 添加 3.333 mL of distilled water when the solution will be used, the unused reagents are stored at 4℃.

描述

琥珀酸脫氫酶 (SDH, 歐共體 1.3.5.1) 廣泛存在於動物體內, 植物, 微生物和培養細胞. SDH is a marker enzyme of mitochondria, which is a membrane binding enzyme located in the inner membrane of mitochondria. It is also one of the key points of respiratory electron transfer and oxidative phosphorylation. 另外, it provides electrons for the respiratory chain of various prokaryotic cells.

SDH can catalyze the dehydrogenation of succinic acid to fumaric acid. The dehydrogenation can reduce 2,6-dichlorophenol indophenol (DCPIP) under the transfer of phenazine dimethyl sulfate (PMS). 2,6- DCPIP has a characteristic absorption peak at 600 奈米. The reduction rate of 2,6-DCPIP is determined by the change of absorbance at 600 奈米, which represents the activity of SDH enzyme.

需要但未提供

分光光度計, 水浴, tabletop centrifuge, 可調式移液器, 研缽/均質器, 1 毫升玻璃比色皿, 冰和蒸餾水.

協定

我. Extraction of SDH:

Accurately weigh 0.1 g of tissue or collect 5 百萬個細胞, 添加 1 mL of Reagent I and 10 µL 試劑 II, homogenize by using homogenizer/mortar in ice bath, fully grind, 離心機在 11000 ×g 為 10 4℃ 分鐘, 取上清液置於冰上進行測試.

二. 程式

1. 預熱分光光度計 30 分分鐘, 調整波長至 600 nm 並用蒸餾水調零.
2. Procedure test

Add each reagent to 1 mL glass cuvette in turn, and start timing at the same time of adding Reagent VI, record the initial absorbance A1 at the wavelength of 600 奈米對於 20 秒. Then put the cuvette together with the reaction solution into a water bath of 37℃ (哺乳動物) 或25℃ (其他物種), and accurate reaction for 1 分分鐘. Quickly take out the cuvette and dry it, and record the absorbance A2 at 80 seconds at 600 奈米. ΔA = A1-A2, obtain ΔAT, ΔAB.

三、. Calculation of SDH activity

Calculation formula for determination with 1 毫升玻璃比色皿.

• 蛋白質濃度

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every milligram tissue protein.

SDH(U/毫克蛋白質)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(心肺復甦×VS) ÷T＝1555.556×(ΔAT-ΔAB)÷心肺復甦

• 樣品重量

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every gram tissue.

SDH(U/g)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(VS÷VST×W) ÷T＝1571.111×(ΔAT-ΔAB)÷W

• Germ or cells

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every 10 thousand germ or cells.

SDH(U/104細胞)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(VS÷VST×500) ÷T

＝3.142×(ΔAT-ΔAB)

虛擬實境測試: 總反應體積, 0.98×10-3 L;

e: The molar extinction coefficient of 2,6-DCPIP, 2.1×104 L/mol/cm; d: The light diameter of cuvette, 1 公分;

VS: 樣品量, 0.03 毫升;

變速: Add the volume of Reagent I and Reagent II, 1.01 毫升; 時間: 反應時間(分分鐘), 1 分分鐘;

心肺復甦: 樣品蛋白濃度, 毫克/毫升; 瓦: 樣品重量, G;

500: Cells or germ, 5 百萬.

筆記

1. All reagents and samples shall be placed on ice during the determination to avoid denaturation and deactivation.
2. 如果ΔA大於 0.5, the enzyme solution should be diluted with enzyme extract to obtain ΔA with less than 0.5, which can improve the detection sensitivity.
3. Because the Extract solution contains a certain concentration of protein (關於 1 毫克/毫升), it is necessary to subtract the protein content of the Extract solution itself when determining the protein concentration of the sample.

實驗範例:

1. Take 0.1g of kidney, 添加 1 mL of Reagent Ⅰ and 10 μL Reagent Ⅱ, grind the homogenate with ice bath, centrifuge at 4℃ and 11000g for 10 分分鐘, and place the supernatant on ice. According to the determination procedure, 酵素活性計算如下: ΔAT= A1T- A2T=0.82-0.681=0.139, ΔAB= A1B- A2B=905-0.904=0.001

SDH activity (U/克質量) = 961.905×(ΔAT- ΔAB) ÷ W =2168.13 U/g mass.

參考

• Fattoretti P, Bertoni-Freddari C, Caselli U, 等人. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging[J]. Mechanisms of aging and development, 1998, 101(1-2): 175- 182.

相關產品

BC0710/BC0715 α-Ketoglutarate Dehydrogenase(α-KGDH) 活性測定試劑盒

BC2150/BC2155 Citric Acid(CA) 含量測定試劑盒

BC0380/BC0385 Pyruvate Dehydrogenase(PDH) 活性測定試劑盒