BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
谷胱甘肽是一种含γ-酰胺键和巯基的三肽，由谷氨酸、半胱氨酸及甘氨酸组成，广泛存在于动、植物组织和微生物中，在生物体内能帮助免疫系统保持正常的功能，并具有抗氧化和整合解毒的作用。谷胱甘肽有还原型（GSH）和氧化型（GSSG）两种形式，GSSG又称为二硫代谷胱甘肽，由两分子谷胱甘肽氧化而成，GSSG会被谷胱甘肽还原酶还原成GSH，因此机体中大多数是以还原型形式存在，还原型与氧化型谷胱甘肽含量的比值（GSH/GSSG）可作为评估细胞氧化还原状态的主要动态指标。
检测原理：通过GSH掩蔽剂抑制样品中原有的GSH，并在谷胱甘肽还原酶催化作用下将GSSG还原为GSH， GSH能够与5,5’-二硫代-双-（2-硝基苯甲酸）（DTNB）反应生成黄色的2-硝基-5-巯基苯甲酸（TNB），产物在412 nm处具有特征吸收峰，通过吸光值变化即可定量检测氧化型谷胱甘肽的含量。
| Component | 48T | Storage |
| 提取液 | 60 mL | 2-8℃. Store in the dark. |
| 试剂一 | 1EA | -20℃. Store in the dark. |
| 试剂二 | 2EA | -20℃ |
| 试剂三 | 2 mL | 2-8℃. Store in the dark. |
| 试剂四 | 28 mL | 2-8℃ |
| 试剂五 | 10 μL | -20℃ |
| 标准品 | 1EA | 2-8℃ |
试剂一（液体1支）：
1、临用前取45μL的试剂一至一支新的EP管中，加1.5mL的乙醇混匀后测定（共配制2支即可）； 2、保存周期与试剂盒有效期相同。
试剂二（EP管2支）：
1、临用前8000g 4℃离心2min使试剂落入管底；
2、加入0.6mL蒸馏水溶解备用。用不完的试剂分装后-20℃保存，禁止反复冻融，一星期内用完。
试剂三（液体1瓶）：
1、若凝固，可在25℃水浴温育片刻至全部融解后使用；
2、保存周期与试剂盒有效期相同。
试剂五（液体1支）：
1、临用前8000g 4℃离心2min使试剂落入管底；
2、加入1.1 mL蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
标准品（粉体1支）：
1、若重新做标曲，则用到该试剂；
2、按照说明书中标曲制作步骤进行配制；
3、溶解后的标品一周内用完。
自备仪器和试剂
研钵（匀浆机）、冰盒（制冰机）、台式离心机、可调式移液枪、水浴锅（烘箱、培养箱、金属浴）、 1ml比色皿、离心管、分光光度计、蒸馏水（去离子水、超纯水均可）。
实验流程
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1、样本提取
1.1 组织样本
称取约0.1g组织，加入1mL提取液，进行冰浴匀浆。4℃×12000rpm离心15min，取上清液，置冰上待测。
注：根据研究需求，可按组织质量（g）：提取液体积(mL)为1：10的比例进行提取。
1.2 细菌/细胞样本
先收集细菌或细胞到离心管内，离心后弃上清；取500万细菌或细胞加入1mL提取液；超声波破碎细菌或细胞（功率300w，超声3秒，间隔7秒，总时间3min）；4℃×12000rpm 离心15min，取上清液，置冰上待测。
注：若增加样本量，可按照细菌/细胞数量（10⁴个）：提取液体积(mL)为500~1000：1的比例提取
1.3 液体样本
直接检测。若浑浊，离心后取上清检测。
2、检测步骤
2.1 可见分光光度计预热30min，调节波长到412nm，蒸馏水调零。
2.2 所有试剂在使用前需在25℃水浴中保温10min。若批量测定则试剂二和三和四可按照 20:40:520 配成混合液，按照580μL加样量操作。
2.3 在1mL玻璃比色皿中依次加入：
| 试剂组分（μL） | 测定管 |
| 样本 | 80 |
| 试剂一 | 40 |
轻轻混匀，孵育10分钟。
| 试剂二 | 20 |
| 试剂三 | 40 |
| 试剂四 | 520 |
| 试剂五 | 20 |
混匀，室温（25℃）下，立即于412nm读取吸光值 A1，25min后读取吸光值A2，ΔA=A2-A1。
注：
(1) 若ΔA在零附近徘徊，可增加样本加样量（如增至160μL），则试剂四相应减少，保持反应总体积720μL不变。
(2) 严格控制反应时间于25min读值。
3、标准曲线的制作
3.1 标准品溶于1.06mL蒸馏水中，（母液需在两天内用且-20℃保存），标准品母液浓度为10 μmol/mL。将母液用蒸馏水稀释成六个浓度梯度的标准品，例如：0、1、2、3、4、5nmol/mL。也可根据实际样本调整标准品浓度。
3.2 标品稀释参照表如下：
吸取标准品母液500μL，加入500μL蒸馏水，混匀得到5μmol/mL的标品稀释液待用。
| 标准品工作液 | 标品稀释液（μL） | 水（μL） | 浓度 (μmol/mL) |
| Std.1 | 0 | 200 | 0 |
| Std.2 | 40 | 160 | 1 |
| Std.3 | 80 | 120 | 2 |
| Std.4 | 120 | 80 | 3 |
| Std.5 | 160 | 40 | 4 |
| Std.5 | 200 | 0 | 5 |
各标准管混匀待用。
3.3 依据测定管的加样表操作，根据结果，以各浓度吸光值减去0浓度吸光值，过0点制作标准曲线。
| 试剂名称（μL） | 标准管 | 0浓度管（仅做一次） |
| 标品 | 80 | |
| 蒸馏水 | 80 | |
| 试剂一 | 40 | 40 |
轻轻混匀，孵育10分钟。
| 试剂二 | 20 | 20 |
| 试剂三 | 40 | 40 |
| 试剂四 | 520 | 520 |
| 试剂五 | 20 | 20 |
混匀，室温（25℃）下，立即于412nm读取吸光值A，ΔA=A测定-0浓度管。
4、结果计算
4.1 标准曲线方程为y=1.0585x-0.0077，R²=0.9985；x为标准品质量（nmol），y为ΔA。
4.2 样本GSSG含量计算
(1) 按样本鲜重计算
GSSG (nmol/g 鲜重)=(ΔA+0.0077)÷1.0585÷(W×V1÷V)=11.8×(ΔA+0.0077) ÷W
(2) 按细胞数量计算
GSSG (nmol/10⁴ cell)=(ΔA+0.0077)÷1.0585÷(细胞数量×V1÷V)=11.8×(ΔA+0.0077)÷细胞数量
(3) 按照液体体积计算
GSSG (nmol/mL)=(ΔA+0.0077)÷1.0585÷V1=11.8×(ΔA+0.0077)
参数说明
V：加入提取液体积，1mL；
V1：反应中加入样本体积，80μL=0.08mL；
W：样品质量，g。
注意事项
1、粗提液不能用于测定可溶性蛋白含量。如需测定蛋白浓度，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
2、一些还原剂如抗坏血酸，巯基乙醇，二硫苏糖醇（DTT）和半胱氨酸，或巯基反应性化合物如马来酰亚胺化合物会干扰谷胱甘肽测定。因此在样品制备过程中应避免使用这些物质。
3、本产品仅供科学研究使用，不适用于临床诊断。
Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamic acid, cysteine, and glycine. It is widely distributed in animal and plant tissues and microorganisms, helping maintain normal immune system function and possessing antioxidant and detoxification capabilities. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG, also known as glutathione disulfide, is formed by the oxidation of two glutathione molecules. GSSG can be reduced back to GSH by glutathione reductase. Consequently, the reduced form predominates in organisms. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing the cellular redox state.
Detection Principle: Endogenous GSH in the sample is masked by a GSH scavenger. Under the catalysis of glutathione reductase, GSSG is reduced to GSH. The newly generated GSH then reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce a yellow-colored 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. The GSSG content is quantitatively determined by measuring the change in absorbance.
| Component | 48T | Storage |
| Extraction Buffer | 60 mL | 2-8℃. Store in the dark. |
| Reagent 1 | 1EA | -20℃. Store in the dark. |
| Reagent 2 | 2EA | -20℃ |
| Reagent 3 | 2 mL | 2-8℃. Store in the dark. |
| Reagent 4 | 28 mL | 2-8℃ |
| Reagent 5 | 10 μL | -20℃ |
| Standard | 1EA | 2-8℃ |
Reagent Preparation:
Reagent 1 (Liquid, 1 vial):
1.Before use, pipette 45 μL of Reagent 1 into a new microcentrifuge tube. Add 1.5 mL of ethanol and mix well for assay (prepare 2 tubes is sufficient).
2.The prepared solution can be stored for the duration of the kit's validity period.
Reagent 2 (Microcentrifuge tubes, 2 vials):
1.Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.
2.Add 0.6 mL of distilled water per vial to dissolve. Use after preparation.
3.Unused dissolved reagent can be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles. Use within one week.
Reagent 3 (Liquid, 1 bottle):
1.If solidified, incubate in a 25°C water bath briefly until completely melted before use.
2.The prepared solution can be stored for the duration of the kit's validity period.
Reagent 5 (Liquid, 1 vial):
1.Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the liquid at the bottom.
2.Add 1.1 mL of distilled water to dissolve. Use after preparation.
3.The prepared solution can be stored for the duration of the kit's validity period.
Standard (Powder, 1 vial):
1.Use this reagent if a new standard curve needs to be prepared.
2.Prepare according to the standard curve preparation steps in the instructions.
3.Use the dissolved standard within one week.
User-Prepared Instruments and Reagents
Mortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 1 mL cuvette, Centrifuge tubes, Spectrophotometer, Distilled water (Deionized water or Ultrapure water are acceptable).
Experimental Procedure
It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C for 15 minutes. Collect the supernatant and keep it on ice for assay.
Note: Depending on research needs, a tissue mass (g) to Extraction Buffer volume (mL) ratio of 1:10 can be used for extraction.
1.2 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Disrupt the bacteria or cells by sonication (power 300W, pulse 3s on, 7s off, total duration 3 min). Centrifuge at 12,000 rpm, 4°C for 15 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1.
1.3 Liquid Samples
Assay directly. If turbid, centrifuge and use the supernatant for assay.
2. Assay Steps
2.1 Preheat the visible spectrophotometer for 30 minutes. Set the wavelength to 412 nm. Zero the instrument with distilled water.
2.2 Incubate all reagents in a 25°C water bath for 10 minutes before use. For batch analysis, Reagents 2, 3, and 4 can be pre-mixed in a ratio of 20:40:520. Use 580 μL of this mixture per assay.
2.3 Add reagents sequentially to a 1 mL glass cuvette:
| Reagent | Volume (μL) - Test Tube |
| Sample | 80 |
| Reagent 1 | 40 |
Mix gently and incubate for 10 minutes.
| Reagent 2 | 20 |
| Reagent 3 | 40 |
| Reagent 4 | 520 |
| Reagent 5 | 20 |
Mix thoroughly. At room temperature (25°C), immediately read the absorbance at 412 nm (A₁). Read the absorbance again after 25 minutes (A₂). Calculate ΔA = A₂ - A₁.
Note:
(1) If ΔA is close to zero, increase the sample volume (e.g., to 160 μL), correspondingly decreasing the volume of Reagent 4 to maintain a total reaction volume of 720 μL.
(2) Strictly control the reaction time, reading A₂ exactly at 25 minutes.
3. Standard Curve Preparation
3.1 Dissolve the Standard in 1.06 mL of distilled water (the stock solution must be used within two days and stored at -20°C). The concentration of the standard stock solution is 10 μmol/mL. Dilute the stock solution with distilled water to create six standard solutions with concentration gradients, e.g., 0, 1, 2, 3, 4, 5 nmol/mL. Standard concentrations can be adjusted based on actual sample levels.
3.2 Standard Dilution Reference Table:
Pipette 500 μL of the standard stock solution and add 500 μL of distilled water. Mix well to obtain a 5 μmol/mL standard dilution for use.
| Standard Working Solution | Volume of Std. Dilution (μL) | Volume of Water (μL) | Concentration (nmol/mL) |
| Std.1 | 0 | 200 | 0 |
| Std.2 | 40 | 160 | 1 |
| Std.3 | 80 | 120 | 2 |
| Std.4 | 120 | 80 | 3 |
| Std.5 | 160 | 40 | 4 |
| Std.5 | 200 | 0 | 5 |
Mix each standard tube well for use.
3.3 Operate according to the assay procedure for the Test tubes. Using the results, subtract the absorbance of the 0 concentration standard from the absorbance of each standard concentration to create a standard curve passing through the origin.
Assay Table for Standard Curve:
| Reagent (μL) | Standard Tube | 0 Conc. Tube (once) |
| Standard | 80 | |
| Distilled Water | 80 | |
| Reagent 1 | 40 | 40 |
Mix gently and incubate for 10 minutes.
| Reagent 2 | 20 | 20 |
| Reagent 3 | 40 | 40 |
| Reagent 4 | 520 | 520 |
| Reagent 5 | 20 | 20 |
Mix thoroughly. At room temperature (25°C), immediately read the absorbance at 412 nm (A). Calculate ΔA = A<sub>Standard Tube</sub> - A<sub>0 Concentration Tube</sub>.
4. Calculation of Results
4.1 Standard Curve Equation: y = 1.0585x - 0.0077, R² = 0.9985; where x is the standard amount (nmol), and y is ΔA.
4.2 Sample GSSG Content Calculation
(1) Based on Sample Fresh Weight
Derived Formula: GSSG (nmol/g fresh weight) = (ΔA + 0.0077) ÷ 1.0585 ÷ (W × V₁ ÷ V)
Simplified Formula: GSSG (nmol/g fresh weight) = 11.8 × (ΔA + 0.0077) ÷ W
(2) Based on Cell Count
Derived Formula: GSSG (nmol/10⁴ cells) = (ΔA + 0.0077) ÷ 1.0585 ÷ (Cell Count × V₁ ÷ V)
Simplified Formula: GSSG (nmol/10⁴ cells) = 11.8 × (ΔA + 0.0077) ÷ Cell Count
(3) Based on Liquid Volume
Derived Formula: GSSG (nmol/mL) = (ΔA + 0.0077) ÷ 1.0585 ÷ V₁
Simplified Formula: GSSG (nmol/mL) = 11.8 × (ΔA + 0.0077)
Parameter Definitions:
V: Volume of Extraction Buffer added (1 mL)
V₁: Volume of sample added to the reaction (80 μL = 0.08 mL)
W: Sample weight (g)
Cell Count: Number of bacteria/cells (in 10⁴)
Precautions
1. The crude extract cannot be used for determining soluble protein content. If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
2. Some reducing agents such as ascorbic acid, β-mercaptoethanol, dithiothreitol (DTT), and cysteine, or thiol-reactive compounds like maleimide compounds, can interfere with the glutathione assay. Therefore, the use of these substances during sample preparation should be avoided.
3. This product is for research use only. Not for use in clinical diagnosis.
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