Hepatic lipase (HL) 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.

操作設備: 分光光度計

目錄號: BC2380

尺寸:50T/24S

成分:

試劑一: 100 毫升×1. 4℃保存.

試劑二: 3 毫升×1. 4℃保存.

試劑三: 粉末×1. 4℃保存. 使用前, 添加 30 毫升蒸餾水, fully dissolve.

試劑四: Powder×2. -20℃保存. 使用前, 添加 2 mL of distilled water to the one, fully dissolve. The dissolved reagent can be stored at -20 °C after repacking. Avoid repeated freeze-thaw cycles;

標準: 粉末×1. 使用前, 6.94 mL of acetone is added to prepare a 10 μmol/mL α-naphthol

標準溶液, which was fully dissolved before use.

產品描述:

Hepatic lipase (HL) is a lipolytic enzyme synthesized in liver parenchymal cells. It is present on the surface of the liver sinusoidal endothelial cells and the surface of the hepatocyte microvilli in the sinusoidal space, and can hydrolyze various lipoproteins. The triglycerides (TG) and phospholipids (PL) in the medium change the size and density of various lipoprotein particles. When the HL and its activity in the plasma increasing, it can lead to low density lipoprotein (LDL) levels in the plasma, increase and accelerate the occurrence and development of atherosclerosis.

HL hydrolyzes α-naphthyl acetate to produce α-naphthol, which can form a purple-red azo compound with fast blue B salt. It has a characteristic absorption peak at 595 奈米, and its color depth is positively correlated with liver esterase activity within a certain range.

需要但未提供的試劑和設備:

分光光度計, 水浴, 平衡, 離心機, adjustable transferpettor, 1 毫升玻璃比色皿, 研缽/均質器, ultrasonic crusher, 冰和蒸餾水.

程式:

我. 酵素 extraction

1. 組織

According to the tissue mass (G): the volume of Reagent I (毫升) 是 1:5~10 to extract. It is recommended to add 1 mL of Reagent I to 0.1 克組織, and fully homogenize on ice bath. Centrifuge at 10000g for 10 4℃分鐘去除不溶物, 並在測試前將上清液放在冰上.

2. 細菌或細胞

According to the bacteria or cells (104): the volume of Reagent I (毫升) 是500~1000:1. It is recommended to add 1 mL of Reagent I to 5 million of bacteria or cells. Use ultrasonication to splitting bacteria and cells (置於冰上, ultrasonic power 300W, 工作時間3秒, 間隔7秒, 總時間 3 分分鐘). 離心機在, 10000克為 10 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

3. Culture medium or other liquid: 直接檢測.

二. 偵測

• 預熱分光光度計 30 分分鐘, 調整波長至 595 奈米, 用蒸餾水調零.
• Preheat reagent III at 30℃ for more than 20 分分鐘.
• 標準: Dilute the 10μmol/mL standard solution to 1.25, 0.625, 0.3125, 0.15625, 0.078μmol/mL with reagent I.
• Add the following reagents in 1.5 mL EP tubes:

三、. 計算:

1.Standard curve

The concentration of standard solution as x-axis, ΔAS as y-axis, obtain the equation y=kx+b. Take ΔAT to the equation to acquire x (微摩爾/毫升) value.

2. 計算

• Tissue protein concentration

單位定義: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every mg of protein in the reaction system per minute at 40℃.

HL Activity (U/毫克蛋白質)=x×Vs÷（Vs×Cpr）÷T =0.1x÷ Cpr

• Tissue weight

單位定義: One unit of enzyme activity is defined as the amount enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every gram of tissue in the reaction system per minute at 40℃.

HL Activity (單位/克重量) = x×Vs÷(W×Vs÷Ve)÷T =0.0333x÷ W

• 液體

單位定義: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every milliliter of liquid sample in the reaction system per minute at 40℃.

HL Activity (單位/毫升) =x× Vs÷Vs÷T=0.1x

• Bacteria or cultured cells

單位定義: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of α-naphthyl acetate to generate 1 μmol of α-naphthol every 104 cells or bacteria in the reaction system per minute at 40℃.

HL Activity (U/104 cell) =x× Ve÷ cell amount÷ T= 0.1x÷ cell amount

VS: 樣品量 (毫升), 0.1 毫升; 維: 萃取液體積, 1 毫升;

心肺復甦: Supernatant sample protein concentration (毫克/毫升); 時間: 反應時間 (分分鐘), 10 分分鐘;

瓦: 樣品重量, G;

細胞數量: 10 thousand as unit.

筆記:

1. If the sample is animal liver, it is recommended to dilute the sample with reagent I more than 25 times before testing, and multiply the dilution factor in the calculation
2. If the sample is serum or plasma from obese animals, it is recommended to dilute the sample with reagent I more than 5 times before testing, and multiply the dilution factor in the calculation
3. When ΔA is greater than 0.8, it is recommended to measure the sample after diluting it with the reagent, and multiply it by the dilution factor in the calculation

實驗範例:

1. 1g rat liver was taken for sample processing, and the supernatant is diluted 24 次, then the operation is carried out according to the operation steps. Measured and calculated by 96 孔板: ΔA = AT-AB = 0.713-0.001=0.712, and the standard curve: y = 0.6381x – 0.0005, calculate x =1.1166

HL activity (U/克質量) = x×VS ÷ (W×VS ÷ VST)÷ T ×48 = 53.597 U/克質量.

2. After the turkey serum was diluted 6 次, the operation was carried out according to the operation steps. Measured and calculated by 96 孔板: ΔA = AT-AB =0.572-0.003=0.569, and the standard curve: y = 0.6381x – 0.0005, calculate x =892

HL activity (U/克質量) = x×VS ÷ (W×VS ÷ VST)÷ T ×12 = 1.071 U/克質量

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