Solarbio氧化型穀胱甘肽 (GSSG) 含量測定試劑盒

氧化型穀胱甘肽 (GSSG) 含量測定試劑盒

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/酶標儀

目錄編號: BC1185

尺寸:100T/96S

成分:

試劑一:100 毫升×1. 4℃保存. 試劑二: 130 微升×1. 4℃保存. 試劑三: 20 毫升×1. 4℃保存. 試劑四:2.5 毫升×1. 4℃保存.

試劑V: Powder ×1. 4℃保存. Dissolve with 2.5 mL of distilled water when the solution will be used, then split into smaller packages, store at -20℃.

試劑六:12.5 微升×1. 4℃保存. Prepare Reagent VI and distilled water according to the sample size at the ratio of 1:20 (V: V) 使用前.

標準: 粉末 10 毫克×1. 4℃保存.

產品描述

氧化型穀胱甘肽(GSSG) is an oxidized form of glutathione (還原型穀胱甘肽), also known as dithioneglutathione, formed by the oxidation of two molecules of glutathione. GSSG is reduced to GSH by glutathione reductase, so most of the body is in the reduced form. The determination of GSH and GSSG content and ratio of GSH/GSSG in cells can reflect the redox status of cells. This kit utilizes reaction of glutathione and 5, 5′-dithiobis-2-nitrobenoic acid (DTNB) to produce 5-thio-2- 硝基苯甲酸. 5-thio-2- nitrobenzoic acid has the largest absorption at wavelength of 412 奈米, and 2-Vinylpyridine inhibit reduced glutathione in the original of samples, and then using glutathione reductase to reduce GSSG to GSH, determining the content of Oxidized Glutathione.

技術規格

最低檢測極限：3.211 微克/毫升

線性範圍：3.9-125 微克/毫升

需要但未提供的試劑和設備.

分析天平, 研缽/均質器, refrigerated centrifuge, 水浴, 可調式移液器, spectrophotometer/ microplate reader, 微型玻璃比色皿/96孔平底板.

程式

我. 樣本 準備

1. 1. 組織樣本

新鮮組織用PBS清洗兩次, 然後加 0.1 g of sample into the homogenizer (均質器在使用前已用試劑 I 沖洗並置於冰上). 添加 1 試劑 I 的毫升數 (the proportion of tissue and reagents can be kept constant), 在冰上充分研磨 (使用液態氮會有更好的研磨效果). 離心機在 8000 ×g and 4℃ for 10 分分鐘, 取上清液置於4℃待測. (The supernatant can be stored at -80℃ for 10 天。)

2. 血液樣本

電漿: 樣品在以下溫度下離心 600 ×g and 4℃ for 10 分分鐘. Absorbing the upper plasma into another tube add with same volume Reagent I. 離心機在 8000 ×g and 4℃ for 10 分分鐘, 取上清液置於4℃待測. (The Supernatant can be stored at -80℃ for 10 天。)

血球:樣品在以下溫度下離心 600 ×g and 4℃ for 10 分分鐘. 丟棄上層血漿, wash with treble volume of PBS for 3 次 (mix blood cell with PBS centrifuge at 600 ×g 為 10 分分鐘), 加入等體積的試劑 I. 混合後, 放置在4℃下 10 分分鐘. 離心機在 8000 ×g 為 10 分分鐘, 取上清液置於4℃待測. (The supernatant can be stored at -80℃ for 10 天。)

3. 細胞樣本

Harvesting cell should not less than 106,then wash with PBS for twice (mix cell with PBS centrifuge at 600×g for 10 分分鐘), mix precipitated cell with the volume of PBS for 3 次. 反覆凍融2-3次 (suggest frozen in liquid nitrogen, 37℃水浴溶解). 離心機在 8000 ×g 為 10 分分鐘, 取上清液置於4℃待測. (The supernatant can be stored at -80℃ for 10 天。)

二. 程式

1. 預熱分光光度計/酵素標儀 30 分分鐘, 調整波長至 412 奈米, 使用蒸餾將計數器設為零
2. Preheat Reagent II in water bath for 30 分分鐘: 37℃ (哺乳動物細胞) 或25℃ (其他物種).
3. The standard dilution: dissolve standard with 1 毫升蒸餾水 (4℃) to a concentration of 10 毫克/毫升. Take suitable solution to prepare the standard of concentration of 125μg/mL、 5微克/毫升、31.25微克/毫升、15.625微克/毫升、7.8125微克/毫升、3.90625μg/mLand0μg/mL (The diluent is a ten-fold diluted Reagent I).

4. 添加 20 μL of diluted standard or sample to 0.5 mL centrifuge tube, 添加 1 µL 試劑 II, incubate at 37 ℃ for 30 minutes after mixing.
5. Make standard curves

After the incubation, 添加 140 μL of Reagent III, 20 µL 試劑 IV, 20μL of Reagent V, 和 2 μL of Reagent VI to the standard tube in sequence. After rapid mixing, the light absorption A1 and A2 of 30 s and 150 s respectively were measured at 412 奈米. Absorbance (A2-A1) is the abscissa (X) and concentration is the ordinate (y), making the standard curve.

添加 140 μL of Reagent III, 20 µL 試劑 IV, 20 μL of Reagent V, 和 2 μLof Reagent VI to the sample tubes in sequence. After rapid mixing, the light absorption A1 and A2 of 30 s and 150 s respectively were measured at 412 奈米, ΔA= A2- A1.

三、. 計算

根據標準曲線, sample ΔA into the formula (X), calculate the sample concentration of y

(μg/ml).

• 蛋白質濃度

GSSH (微克 / mg prot)=y×Vrv÷Vrv÷Cpr =y÷Cpr

• 樣品重量

GSSH (μg /g)= y×Vrv÷(Vrv÷Vsv×W)= y÷W

• 賽拉蒙

GSSH (微克 /106 細胞)= y×Vrv÷(Vrv÷Vsv×N)= y÷N

• Solution volume

GSSH (微克 / 毫升)= y×Vrv/Vs= 2y

氮: 細胞數量,106；

繩索: 總反應體積, 0.203 毫升；

電壓: The volume of supernatant was added into the reaction system, 20 微升=0.02毫升； 瓦: 樣品重量, G;

心肺復甦: 上清液蛋白濃度, 毫克/毫升.

筆記:

1. 樣品需要完全均質. 如果暫時無法完成測試, 可在-80℃保存.
2. If the content of GSSG content in the sample is uncertain, several gradients can be diluted before measurement.
3. This method uses the enzymatic reaction rate to calculate the substrate concentration and complete readings as accurately as possible at 30 和 150
4. Reagent I contained protein precipitant, the supernatant could not be used for protein concentration determination. 如果需要測定蛋白質含量, 拿另一張紙巾.

參考:

• 阿爾珀特J, 吉爾伯特·H·F. 透過回收柱後反應檢測氧化型和還原型穀胱甘肽[J]. 分析生物化學, 1985, 144(2):553-562.
• 歐文斯 C W I, Belcher R A colorimetric micro-method for the determination of glutathione[J]. 生化雜誌, 1965, 94(3): 705.

相關產品：

BC1170/ BC1175 Reduced Glutathione（GSH）Assay Kit

BC1190/ BC1195 穀胱甘肽過氧化物酶測定試劑盒

BC0350/ BC0355 Glutathione S-transferase(GST) 活性測定試劑盒

BC1160/ BC1165 Glutathione Reductase (GR) 檢測試劑盒