Hepatic lipase (HL) Набор для анализа активности

Примечание: Перед тестированием возьмите два или три разных образца для прогнозирования..

Операционное оборудование: Спектрофотометр

Кот нет: BC2380

Размер:50Т/24С

Компоненты:

Реагент I: 100 мл×1. Хранение при 4℃.

Реагент II: 3 мл×1. Хранение при 4℃.

Реагент III: Порошок×1. Хранение при 4℃. Перед использованием, добавлять 30 mL of distilled water, fully dissolve.

Реагент IV: Порошок×2. Хранение при -20℃. Перед использованием, добавлять 2 mL of distilled water to the one, fully dissolve. The dissolved reagent can be stored at -20 °C after repacking. Избегайте повторяющихся циклов замораживания-оттаивания;

Стандартный: Порошок×1. Перед использованием, 6.94 mL of acetone is added to prepare a 10 μmol/mL α-naphthol

standard solution, which was fully dissolved before use.

Описание продукта:

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 (ТГ) 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 нм, and its color depth is positively correlated with liver esterase activity within a certain range.

Требуемые реагенты и оборудование, которые не входят в комплект поставки:

Спектрофотометр, водяная баня, баланс, центрифуга, adjustable transferpettor, 1 стеклянная кювета мл, ступка/гомогенизатор, ultrasonic crusher, лед и дистиллированная вода.

Процедура:

я. Фермент extraction

1. Салфетка

According to the tissue mass (г): the volume of Reagent I (мл) is 1:5~10 to extract. It is recommended to add 1 mL of Reagent I to 0.1 г ткани, and fully homogenize on ice bath. Centrifuge at 10000g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.

2. Бактерии или клетки

According to the bacteria or cells (104): the volume of Reagent I (мл) is 500~1000:1. It is recommended to add 1 mL of Reagent I to 5 миллионов бактерий или клеток. Use ultrasonication to splitting bacteria and cells (помещенный на лед, ultrasonic power 300W, working time 3s, интервал 7 секунд, общее время 3 мин). Центрифуга в, 10000g for 10 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

3. Culture medium or other liquid: Обнаружение напрямую.

II. Обнаружение

• Предварительно разогрейте спектрофотометр для 30 минуты, отрегулировать длину волны, чтобы 595 нм, установить ноль с помощью дистиллированной воды.
• Preheat reagent III at 30℃ for more than 20 минуты.
• Стандартный: 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:

III. Расчет:

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 (мкмоль/мл) value.

2. Расчет

• Tissue protein concentration

Определение единицы измерения: 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 (Ед/мг прот)=x×Vs÷（Vs×Cpr）÷T =0.1x÷ Cpr

• Tissue weight

Определение единицы измерения: 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 (Е/г веса) = x×Vs÷(W×Vs÷Ve)÷T =0.0333x÷ W

• Жидкость

Определение единицы измерения: 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 (Ед/мл) =x× Vs÷Vs÷T=0.1x

• Bacteria or cultured cells

Определение единицы измерения: 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

Против: Объем образца (мл), 0.1 мл; ве: Extract solution volume, 1 мл;

КПР: Supernatant sample protein concentration (мг/мл); Т: Время реакции (мин), 10 минуты;

Вт: Вес образца, г;

Количество ячеек: 10 thousand as unit.

Примечание:

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

Экспериментальный пример:

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

HL activity (Ед/г массы) = x×VS ÷ (W×VS ÷ VST)÷ T ×48 = 53.597 Ед/г массы.

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

HL activity (Ед/г массы) = x×VS ÷ (W×VS ÷ VST)÷ T ×12 = 1.071 Ед/г массы

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