Соларбио Гидроксипролин (ГИП) Набор для анализа активности

Гидроксипролин (ГИП) Набор для анализа активности

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

Оборудование обнаружения: Спектрофотометр/считыватель микропланшетов

Кот нет: BC0255

Размер: 100Т/96С

Компоненты:

Экстракт раствора: 6 mol/L hydrochloric acid (HCl), self-provided reagent. Concentrated HCl（ 37%） : H2O(V/V) =1:1, stored at room temperature.

Реагент I: 8 мл×1, store at 4℃ and protect from light. Реагент II: 8 мл×1, store at 4℃ and protect from light. Стандартный: 0.5 мл×1, 0.5 mg/mL hydroxyproline, хранить при 4℃.

Описание:

HYP is one of the main components of collagen in the body. Most of the collagen is distributed in the skin, tendon, cartilage, and blood vessels et al. Поэтому, the content of HYP is an important index reflecting the metabolism and fibrosis degree of collagen tissue.

The sample is hydrolyzed to produce free HYP, which is further oxidized by chloramine T. The oxidized product reacted with p-Dimethylaminobenzaldehyde to produce a red compound with characteristic absorption peak at 560 нм. The content of HYP can be calculated by measuring the absorption value of sample hydrolysate at 560 нм.

Требуется, но не предусмотрено:

Scales, oven, glass tube, центрифуга, водяная баня, spectrophotometer/microplate reader, micro glass cuvette/96-well plate, 6 mol/L HCl and distilled water.

Процедура:

я. Базовые приготовления

1. 1. Образец ткани:

Weigh about 0.2 g of the sample into the glass tube, cut the tissue into pieces as much as possible for digestion. Добавлять 2 mL of Extract solution and the cover is slightly loose and not airtight, boil it or bake it in 110℃ oven for 2 к 6 hours to digest it until there is no visible big lump.

После охлаждения, adjust the pH to 6-8 with 10 mol/L NaOH (Do not over acid or over alkali) then constant volume to 4 mL with distilled water. Centrifugation at 16000 об/мин для 20 minutes at 25℃ (if there is still impurity after centrifugation, it can be removed by filtration).

Take the supernatant for test. (Black substance may be formed in the process, and if it cannot be digested for a long time, it may be carbonized substance. It does not affect the experiment).

2. Клетки:

Брать 5 миллион клеток, добавлять 1 mL of Extract solution, boil, or oven at 110℃ for 2 к 6 hours to digest to transparent state.

После охлаждения, adjust the pH to 6-8 with 10 mol/L NaOH (Do not over acid or over alkali) then constant volume to 2 mL with distilled water. Centrifugation at 16000 об/мин для 20 minutes at 25℃ (if there is still impurity after centrifugation, it can be removed by filtration).

Take the supernatant for test.

II. Обнаружение

1. Предварительный нагрев спектрофотометра/считывателя микропланшетов для 30 минуты, отрегулировать длину волны, чтобы 560 nm and set zero with distilled
2. Dilute the standard solution to 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234 μg/mL.
3. Add reagents as the following table.

Хорошо перемешать, incubate at 60℃ for 20 минуты, leave it at room temperature for 15 минуты, take 200μL of the reaction solution into micro glass cuvette/96 well flat-bottom plate and test the absorbance value of at 560 нм. ΔA = AT – АБ.

III. Расчет

1. Making of standard

When making the standard curve, the concentration of the standard solution is taken as the x-axis, and the ΔAS (ΔAS =AS-AB) is taken as the y-axis. The linear equation y=kx+b is obtained. Take ΔAT (AT-AB) to the equation to acquire x.

2. Calculation of hydroxyproline content:calculated according to the fresh weight of the sample:

Tissue hydroxyproline content (μg/g fresh weight) = x×VS÷(W×VS÷VTE) = 4x÷W.

• Calculated according to the sample protein concentration:

Tissue hydroxyproline content (μg/mg prot) = x×VS÷(Цпр×ВС) = x÷Cpr.

• Calculated according to the number of bacteria orcells:

Cell hydroxyproline content (μg/104 cell) = x×VS÷(N×VS÷VCE )= 2x÷N.

ПРОТИВ: Volume of added sample, 0.06 мл;

VTE: Volume of tissue extract solution, 4 мл; VCE: Volume of cell extract solution, 2 мл;

Вт: Fresh weight of sample, г;

Н: Number of cells,104 as units, 10000;

КПР: Concentration of sample protein, мг/мл.

Примечание

1. If the OD value is greater than 1.0, the sample should be diluted properly and then determined. Pay attention to multiply the dilution multiple in the calculation formula.
2. The reagent has certain toxicity. Please take protective measures during operation to prevent inhalation or contact with skin.
3. When calculating according to the sample protein concentration, the protein in the sample itself needs to be extracted separately and determined.

Экспериментальные примеры

• 0.2g of mouse leg is taken for sample treatment, and the supernatant is taken out and operated according to the determination steps. ΔA =AT-AB = 0.177-0.06 "=" 0.117 is measured by 96 колодезная пластина, and the standard curve y = 0.0383x + 0.022 is brought in, and x = 2.48 is

The content of hydroxyproline in tissue (μg/g mass) = 4x ÷ W = 4 × 2.48 ÷ 0.2 "=" 49.6 μg/g mass.

Recent Product Citations

• Litong Fan,Jiaqing Chen,Yanmeng Tao,и другие. Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin. Journal of Orthopaedic Research. January 2019;(IF3.043)

сопутствующие товары

BC1550/BC1555 Glutamic-pyruvic Transaminase (GPT) Activity Assay Kit BC1560/BC1565 Glutamic-oxalacetic Transaminase (GOT) Activity Assay Kit BC0290/BC0295 Proline (PRO) Набор для анализа содержания

BC1570/BC1575 Amino Acid (AA) Content Assay Kit BC0180/BC0185 Cysteine (Cys) Content Assay Kit BC1580/BC1585 Glutamic Acid (Glu) Набор для анализа содержания

Технические характеристики:

Detection Limit: 0.057 μg/mL

Линейный диапазон: 0.117-30 μg/mL