肝リパーゼ (HL) 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: 分光光度計

カタログ番号: BC2380

サイズ:50T/24S

コンポーネント:

試薬I: 100 mL×1. 4℃で保存.

試薬II: 3 mL×1. 4℃で保存.

試薬Ⅲ: パウダー×1. 4℃で保存. ご使用の前に, 追加 30 蒸留水 mL, fully dissolve.

試薬IV: パウダー×2. -20℃で保管. ご使用の前に, 追加 2 mL of distilled water to the one, fully dissolve. The dissolved reagent can be stored at -20 °C after repacking. 凍結融解サイクルを繰り返さないようにする;

標準: パウダー×1. ご使用の前に, 6.94 mL of アセトン is added to prepare a 10 μmol/mL α-naphthol

標準液, which was fully dissolved before use.

製品説明:

肝リパーゼ (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 nm, and its color depth is positively correlated with liver esterase activity within a certain range.

必要だが提供されていない試薬と装置:

分光光度計, 水浴, バランス, 遠心, adjustable transferpettor, 1 mLガラスキュベット, モルタル/ホモジナイザー, ultrasonic crusher, 氷と蒸留水.

手順:

私. 酵素 抽出

1. 組織

According to the tissue mass (g): the volume of Reagent I (mL) は 1:5~10 to extract. It is recommended to add 1 mL of Reagent I to 0.1 組織グラム, and fully homogenize on ice bath. Centrifuge at 10000g for 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. 細菌とか細胞とか

According to the bacteria or cells (104): the volume of Reagent I (mL) 500~1000です:1. It is recommended to add 1 mL of Reagent I to 5 数百万の細菌または細胞. 超音波を使用して細菌と細胞を分割します。 (氷の上に置かれた, ultrasonic power 300W, 作業時間3秒, インターバル7秒, 合計時間 3 分). で遠心分離します。, 10000グラムのための 10 4℃で3分間放置して不溶性物質を除去し、試験前に上清を氷上に取ります。.

3. Culture medium or other liquid: 直接検出.

Ⅱ. 検出

• 予熱用分光光度計 30 分, 波長を調整して 595 nm, 蒸留水でゼロを設定する.
• Preheat reagent III at 30℃ for more than 20 分.
• 標準: Dilute the 10μmol/mL standard solution to 1.25, 0.625, 0.3125, 0.15625, 0.078μmol/mL with reagent I.
• 次の試薬を追加します。 1.5 mL EP tubes:

	Contrast tube (C)	試験管 (T)	標準チューブ (S)	ブランクチューブ (B)
サンプル (μL)	100	100	–	–
標準溶液 (μL)	–	–	100	–
試薬I (μL)	450	400	400	500
試薬II (μL)	–	50	50	50
Mix and react for 10 min at 30℃			–	–
試薬Ⅲ (μL)	400	400	400	400
試薬IV (μL)	50	50	50	50
Mix thoroughly and detect the absorbance at 595 nm, record as AC, で, AS and AB respectively. ΔAT=(AT-AC), ΔAS=(as-ab). A contrast tube is required for each test tube, and the standard curve need only be tested once or twice.

Ⅲ. 計算:

1.標準曲線

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 (μmol/mL) 価値.

2. 計算

• 組織タンパク質濃度

単位の定義: 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/mgプロット)=x×Vs÷（Vs×Cpr）÷T =0.1x÷ Cpr

• 組織重量

単位の定義: 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 (U/g重量) = x×Vs÷(W×Vs÷Ve)÷T =0.0333x÷ W

• 液体

単位の定義: 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 (U/mL) =x× Vs÷Vs÷T=0.1x

• 細菌または培養細胞

単位の定義: 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セル) =x× Ve÷ cell amount÷ T= 0.1x÷ cell amount

対: サンプル量 (mL), 0.1 mL; ヴェ: 抽出液量, 1 mL;

心肺蘇生法: 上清サンプルのタンパク質濃度 (mg/mL); T: 反応時間 (分), 10 分;

W: サンプル重量, g;

細胞量: 10 thousand as unit.

注記:

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

実験例:

1. 1g rat liver was taken for sample processing, and the supernatant is diluted 24 回, then the operation is carried out according to the operation steps. Measured and calculated by 96 ウェルプレート: 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/g質量) = x×VS ÷ (W×VS ÷ VST)÷ T ×48 = 53.597 U/g質量.

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

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

関連製品：

BC2340/BC2345 Lipase(LPS) 活性アッセイキット

BC0620/BC0625 Triglyceride(TG) コンテンツアッセイキット

BC2440/BC2445 Lipoprteinlipase(LPL) 活性アッセイキット

BC0320/BC0325 Plant Lipoxygenase(LOX) 活性アッセイキット

BC0590/BC0595 Free fatty Acids(FFA) コンテンツアッセイキット