Solarbio Восстановленный глутатион (ГШ) Набор для анализа содержания

Уменьшенный глутатион (ГШ) Набор для анализа содержания

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

Операционное оборудование: Spectrophotometer/Microplate Reader

Номер каталога: BC1175

Размер: 100Т/96С

Компоненты:

Реагент I: 100 мл×1. Хранить при 4℃.

Реагент II: 20 мл×1. Хранить при 4℃.

Реагент III: 8 мл×1. Хранить при 4℃, protect from light.

Стандартный: Пудра 10 mg×1. Хранить при 4℃, protect from light.

Описание продукта

Glutathione is a natural tripeptide composed of glutamic acid (Glu), cysteine (Cys) and glycine (Gly). It is a kind of compound containing sulfhydryl group (-Ш.Х.), which widely exists in animal tissue, растительная ткань, microorganism, and yeast. Glutathione can react with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (ГССГ). 2-nitro-5-mercaptobenzoic acid is a yellow product, with the maximum absorption at 412 нм.

Технические характеристики

Minimum Detection Limit：3.763 μg/mL

Линейный диапазон：12.5-400 μg/mL

Требуемые реагенты и оборудование, которые не входят в комплект поставки

Analytical balance, ступка/гомогенизатор, low temperature centrifuge, водяная баня, регулируемая пипетка, spectrophotometer/microplate reader, micro glass cuvette or 96 well flat-bottom plate and distilled water.

Процедура

я. Базовые приготовления

1. Образец ткани

Wash fresh tissues with PBS for twice, затем добавьте 0.1 g of animal/plant tissue into homogenizer (the homogenizer has been rinsed with Reagent I and placed on ice before use). Добавлять 1 мл реагента I (the proportion of tissue and Reagents can be kept constant), полностью шлифовать на льду (using liquid nitrogen will have a better grinding effect). Центрифуга в 8000 ×g для 10 минут при 4℃, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

2. Образец крови

Плазма: Sample is centrifuged at 600 ×g для 10 минут при 4℃. Absorbing the upper plasma into another tube with adding the same volume Reagent I. Центрифуга в 8000 ×g для 10 минут при 4℃, take the supernatant and place it at 4℃for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

Blood cell: Sample is centrifuged at 600 ×g для 10 минут при 4℃. Discarding the upper plasma, wash with three times volume of PBS for 3 раз (re-suspend blood cell with PBS, центрифуга в 600 ×g для 10 минуты), add equal volume of Reagent I. After mixing, it is placed at 4℃ for 10 минуты. Центрифуга в 8000 ×g для 10 минуты, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

3. Клетка образец

Harvesting cell should not less than 106,then wash it with PBS for twice (re-suspend cell with PBS, центрифуга в 600 ×g для 10 минуты). The volume of Reagent I added is three times the volume of cell precipitation to re-suspend the cells. Repeated freezing and thawing 2–3 times (It is suggested that frozen in liquid nitrogen, dissolved in 37℃ water bath). Центрифуга в 8000 ×g для 10 минуты, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

II. Процедура

1. Предварительный нагрев спектрофотометра/считывателя микропланшетов для 30 минуты, отрегулировать длину волны, чтобы 412 нм, set zero with distilled
2. Preheat Reagent II in 37℃ (mammal cell) или 25℃ (другие виды) водяная баня для 30
3. Blank tube determination: take micro glass cuvette, добавлять 20 мкл дистиллированной воды, 140 мкл реагента II, 40 μL of Reagent III in turn, хорошо перемешать, place for 2 минуты, and measure 412 nm absorbance AB.
4. Making standard curve

Weigh 1 mg of standard and dissolve it with 1 mL of distilled water to obtain the concentration of 1 мг/мл. Take the appropriate solution to prepare the standards with the concentration of 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL (Dilute Reagent I ten times before diluting the standard solution).

Take a 1.5 mL EP tube and add 20 μL of standard, 140 μL of Reagent II and 40 μL of Reagent III in turn. After each tube is evenly mixed, it is allowed to stand for 2 минуты. Измерьте поглощение при 412 нм, and absorbance minus AB as abscissa. Make the standard curve according to the absorbance (Икс) and concentration (й, μg/mL).

5. Sample tube test: take micro glass cuvette, добавлять 20 μL of sample, 140 мкл реагента II, 40 μL of Reagent III in turn, хорошо перемешать, and then stand for 2 minutes to test the absorbance ATat 412 нм, ΔA = AT – АБ.
6. The operation of the microplate reader is the same as that of the spectrophotometer, and the operation is as fast as

III. Расчеты

According to the standard curve, take sample ΔA into the formula(Икс), and calculate the sample concentration y (μg/mL).

• Концентрация белка

ГШ (μg/mg prot)=y×VRV÷VRV÷Cpr =y÷Cpr

• Вес образца

ГШ (μg/g)=y×VRV÷(VRV÷VSV×W)= y÷W

• Количество ячеек

ГШ (μg/104cell)=y×VRV÷(VRV÷VSV×N)= y÷N

• Solution volume GSH (μg/mL)=2y

Н: Количество ячеек, 106;

VSV: Total supernatant volume, 1 мл；

VRV: Supernatant volume added into the reaction system, 20 μL=0.02 mL； Вт: Вес образца, г;

КПР: Концентрация белка в супернатанте, мг/мл.

2: The volume of plasma (blood cells) is diluted by one time.

Примечание:

1. The sample needs to be homogenized completely. If the test cannot be completed temporarily, it can be stored at-80℃.
2. Стандартный: Reduced glutathione is prepared when the solution will be
3. If the GSH content in the sample is uncertain, Dilute the sample for several gradients before
4. Because Reagent I contain protein precipitant, the supernatant cannot be used for protein concentration determination. If the protein content needs to be determined, take another tissue.

Ссылка:

• Alpert A J, Gilbert H F. Detection of oxidized and reduced glutathione with a recycling postcolumn reaction[Дж]. Analytical biochemistry, 1985, 144(2):553-562.
• Owens C W I, Belcher R V. A colorimetric micro-method for the determination of glutathione[Дж]. Biochemical Journal, 1965, 94(3):

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