糖原是由葡萄糖构成的高分子多糖，是糖主要的储存形式之一，主要贮存在肝和肌肉中作为备用能量，分别称为肝糖原和肌糖原。肝糖原可调节血糖浓度，当血糖升高时可在肝脏合成糖原，血糖降低时，肝糖原则分解为葡萄糖以补充血糖。因此，肝糖原对维持血糖的相对平衡十分重要。肌糖原是肌肉中糖的储存形式，在剧烈运动消耗大量血糖时，肌糖原不能直接分解成血糖，必须先分解产生乳酸，随血液循环到肝脏，通过糖异生转变为肝糖原和葡萄糖。
本试剂盒的检测原理为：利用强碱性提取液提取糖原，在强酸性条件下与蒽酮显色剂形成在620 nm处有特征吸收峰的蓝色化合物，在一定的浓度范围内，糖原含量与620 nm吸光度成线性关系，根据标准曲线，即可计算出样品中糖原的含量。
产品组分
G1522862 | 组件 | 物理外观 | 100 T | 储存 | 单次测试使用量 |
G1522862A | 碱性提取液 | 液体 | 100 mL | 4℃ | 0.75 mL |
G1522862B | 显色剂 | 粉末 | 4支 | 4℃.避光 | 200 μL |
G1522862C | 葡萄糖标准品 (1 mg/mL) | 液体 | 2 mL | 4℃.避光 | 100 μL |
使用方法
1. 试剂准备
（1）将碱性提取液平衡至室温，该试剂有腐蚀性 ，操作时请做好防护措施。
（2）取1支显色剂，向其中加入5 mL的浓硫酸，充分溶解后避光保存。该溶液在4℃的有效期为3天，配置后请尽快使用。
2、标准曲线设置
按下表所示用去离子水将1 mg/mL标准品稀释为0.25、0.1、0.05、0.025、0.0125、0.00625、0.003125 mg/mL的标准溶液。葡萄糖标准品为无菌状态，开封后在4℃存放易污染。首次使用请在超净工作台打开并进行无菌分装放于4℃。若不能保证无菌状态可分装冻于-20℃。
No. | Standard Volume (μL) | Water (μL) | Concentration (mg/mL) |
Std.1 | 100 μL 1 mg/mL | 300 μL | 0.25 |
Std.2 | 160 μL of Std.1 | 240 μL | 0.1 |
Std.3 | 200 μL of Std.2 | 200 μL | 0.05 |
Std.4 | 200 μL of Std.3 | 200 μL | 0.025 |
Std.5 | 200 μL of Std.4 | 200 μL | 0.0125 |
Std.6 | 200 μL of Std.5 | 200 μL | 0.00625 |
Std.7 | 200 μL of Std.6 | 200 μL | 0.003125 |
3、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80°C保存1个月。
（1）组织样本
称取0.1 g样本，置于10 mL试管中，加入0.75 mL碱性提取液，置于沸水浴中煮沸20 min（塞紧试管塞，防止水分蒸发），每5 min振摇试管1次，充分混匀。待组织全部溶解后，取出试管冷却后去离子水定容到5 mL，混匀，8,000g，25°C离心10 min，取上清液待测。
（2）细胞或细菌样本
收集500万细菌或细胞到EP管内，1×PBS清洗后离心后弃上清，加入0.75 mL碱性提取液超声波破碎细菌或细胞（功率200W，超声3s，间隔10s，重复30次）；转移至10 mL试管中，置于沸水浴中煮沸20 min（塞紧试管塞，防止水分蒸发），每5 min振摇试管1次，充分混匀。取出试管冷却后去离子水定容到5mL，混匀，8,000g，25°C离心10 min，取上清液待测。
4. 样本检测
在管中分别加入下列试剂：
试剂 | 空白管（μL） | 标准管（μL） | 测定管（μL） |
样本 | 0 | 0 | 100 |
标准液 | 0 | 100 | 0 |
去离子水 | 100 | 0 | 0 |
显色剂 | 200 | 200 | 200 |
混匀后置于沸水浴中反应5 min（盖紧，防止水分蒸发），流水冷却后取200 μL转移至96孔板中，读取620 nm波长处测定吸光值，分别记为A空白 ，A标准，A测定。
计算ΔA 测定=A测定-A空白，ΔA标准=A标准-A空白。注意：实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测定小于0.001可适当加大样本量。如果ΔA测定超出标曲范围，样本可用去离子水进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
5. 结果计算
以标准液浓度为X轴，ΔA标准为Y轴，绘制标准曲线。将ΔA测定代入Y计算出X (mg/mL)。
提取液中的总葡萄糖量(mg)=X×V样×（V样总÷V样）×n=X×V样总×n
提取液中总糖原量(mg)=X×n×V样总÷1.11
(1)按照样本质量计算
糖原(mg/g) =（X×n×V样总÷1.11）÷W
(2)按照样本蛋白浓度计算
糖原(mg/mgprot)=（X×n×V样总÷1.11）÷（Cpr×V样总）
(3) 按照细菌或细胞数量计算
糖原(mg/10⁴cells)=（X×n×V样总÷1.11）÷N
参数说明：
1.11：是此法测得葡萄糖含量换算为糖原含量的常数，即100 μg糖原用蒽酮显色试剂显色的颜色相当于111 μg葡萄糖用蒽酮试剂显色的颜色；V样：加入反应体系中待测样本体积，0.1 mL； W：样本质量，g；V样总：样本总体积，5 mL；n：稀释倍数；Cpr：样本蛋白质浓度，mg/mL；细菌或细胞数量：以10⁴为单位，万个。
6. 结果展示
标准曲线
注意事项
1. 正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2. 浓硫酸的浓度必须在95%-98%的分析纯且不可开瓶太久。
3. 配制显色液时先将粉剂导入玻璃容器中，然后加少量浓硫酸，小心搅拌使其溶解，然后再加入剩余的浓硫酸。配置好的显色液放于4℃存放，但不可超过3天，建议现配现用。
4. 葡萄糖标准品为无菌状态，开封后在4℃存放易污染。首次使用请在超净工作台打开并进行无菌分装放于4℃。若不能保证无菌状态可分装冻于-20℃。
5. 本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Glycogen is a high-molecular-weight polysaccharide composed of glucose units and is one of the main storage forms of glucose. It is mainly stored in the liver and muscles as reserve energy, referred to as liver glycogen and muscle glycogen, respectively. Liver glycogen regulates blood glucose concentration: when blood glucose rises, it is synthesized into glycogen in the liver; when blood glucose decreases, liver glycogen is broken down into glucose to supplement blood glucose. Therefore, liver glycogen is very important for maintaining the relative balance of blood glucose. Muscle glycogen is the storage form of glucose in muscles. During strenuous exercise that consumes a large amount of blood glucose, muscle glycogen cannot be directly broken down into blood glucose; it must first be broken down into lactate, which is transported through the bloodstream to the liver, where it is converted into liver glycogen and glucose via gluconeogenesis.
The detection principle of this kit is as follows: Glycogen is extracted using a strong alkaline extraction buffer. Under strong acidic conditions, it reacts with the anthrone chromogenic reagent to form a blue compound with a characteristic absorption peak at 620 nm. Within a certain concentration range, the glycogen content is linearly related to the absorbance at 620 nm. Based on the standard curve, the glycogen content in the sample can be calculated.
Components
G1522862 | Components | Appearance | 100 T | Storage | Quantity Per Test |
G1522862A | Alkaline Extraction Buffer | Liquid | 100 mL | 4°C | 0.75 mL |
G1522862B | Chromogenic Reagent | Powder | 4 tubes | 4°C, Store in the dark. | 200 μL |
G1522862C | Glucose Standard (1 mg/mL) | Liquid | 2 mL | 4°C, Store in the dark. | 100 μL |
Usage Protocol
1. Reagent Preparation
1) Equilibrate the Alkaline Extraction Buffer to room temperature. This reagent is corrosive; please take appropriate protective measures during handling.
2) Take one vial of Chromogenic Reagent, add 5 mL of concentrated sulfuric acid, dissolve thoroughly, and store protected from light. This solution is valid for 3 days at 4 °C; use it as soon as possible after preparation.
2. Standard Curve Setup
Dilute the 1 mg/mL standard with deionized water according to the table below to prepare standard solutions of 0.25, 0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.003125 mg/mL. The glucose standard is provided sterile. Once opened, storage at 4 °C may lead to contamination. For first use, open it in a clean bench and dispense aseptically before storing at 4 °C. If sterility cannot be guaranteed, aliquot and freeze at –20 °C.
No. | Standard Volume (μL) | Water (μL) | Concentration (mg/mL) |
Std.1 | 100 μL 1 mg/mL | 300 μL | 0.25 |
Std.2 | 160 μL of Std.1 | 240 μL | 0.1 |
Std.3 | 200 μL of Std.2 | 200 μL | 0.05 |
Std.4 | 200 μL of Std.3 | 200 μL | 0.025 |
Std.5 | 200 μL of Std.4 | 200 μL | 0.0125 |
Std.6 | 200 μL of Std.5 | 200 μL | 0.00625 |
Std.7 | 200 μL of Std.6 | 200 μL | 0.003125 |
3. Sample Preparation
Note: Fresh samples are recommended. If the experiment is not performed immediately, samples can be stored at –80 °C for one month.
1) Tissue Samples
Weigh 0.1 g of sample, place it into a 10 mL test tube, add 0.75 mL of Alkaline Extraction Buffer, and boil in a boiling water bath for 20 min (cap the test tube tightly to prevent water evaporation). Shake the tube once every 5 min to mix thoroughly. After the tissue is completely dissolved, remove the tube, cool it, and add deionized water to a final volume of 5 mL. Mix well, centrifuge at 8,000 × g for 10 min at 25 °C, and collect the supernatant for testing.
2) Cell or Bacterial Samples
Collect 5 × 10⁶ bacteria or cells into an EP tube, wash with 1× PBS, centrifuge, and discard the supernatant. Add 0.75 mL of Alkaline Extraction Buffer and disrupt the bacteria or cells by ultrasonication (power 200 W, sonicate for 3 s, pause for 10 s, repeat 30 times). Transfer the mixture to a 10 mL test tube, boil in a boiling water bath for 20 min (cap the test tube tightly to prevent water evaporation), shaking once every 5 min to mix thoroughly. Remove the tube, cool it, and add deionized water to a final volume of 5 mL. Mix well, centrifuge at 8,000 × g for 10 min at 25 °C, and collect the supernatant for testing.
4. Sample Detection
Add the following reagents into an EP tube:
Reagent | Blank tube (μL) | Standard tube (μL) | Sample tube (μL) |
Sample | 0 | 0 | 100 |
Standard solution | 0 | 100 | 0 |
Deionized water | 100 | 0 | 0 |
Chromogenic reagent | 200 | 200 | 200 |
Mix well, then react in a boiling water bath for 5 min (cap tightly to prevent water evaporation). Cool under running water, then transfer 200 μL to a 96‑well plate. Measure the absorbance at 620 nm, recorded as A blank, A standard, and A sample respectively.
Calculate ΔAsample = A sample – A blank, ΔA standard = A standard – A blank.
Note: It is recommended to perform a preliminary experiment using 2‑3 samples with expected large differences before formal testing. If ΔA sample is less than 0.001, the sample volume can be appropriately increased. If ΔA sample exceeds the range of the standard curve, the sample can be further diluted with deionized water and the result multiplied by the dilution factor, or the amount of sample used for extraction can be reduced.
5. Result Calculation
Plot the standard curve with the standard concentration as the X‑axis and ΔA standard as the Y‑axis. Substitute ΔA sample into Y to obtain X (mg/mL).
Total glucose content in the extract (mg) = X × V sample × (V total ÷ V sample) × n = X × V total × n
Total glycogen content in the extract (mg) = X × n × V total ÷ 1.11
1) Calculated by sample mass:
Glycogen (mg/g) = (X × n × V total ÷ 1.11) ÷ W
2) Calculated by sample protein concentration:
Glycogen (mg/mg prot) = (X × n × V total ÷ 1.11) ÷ (Cpr × V total)
3) Calculated by bacterial or cell number:
Glycogen (mg/10⁴ cells) = (X × n × V total ÷ 1.11) ÷ N
Parameter description:
1.11: constant for converting glucose content measured by this method to glycogen content, i.e., the color intensity of 100 μg glycogen with anthrone reagent is equivalent to that of 111 μg glucose with anthrone reagent; V sample: volume of the test sample added to the reaction system, 0.1 mL; W: sample mass, g; V total: total sample volume, 5 mL; n: dilution factor; Cpr: protein concentration of the sample, mg/mL; N: number of bacteria or cells, in units of 10⁴.
6. Results Presentation
Standard curve:
Precautions
1. Before formal testing, it is recommended to perform a preliminary experiment using 2‑3 samples with expected large differences.
2. The concentration of concentrated sulfuric acid must be 95%‑98% analytical grade and the bottle should not have been opened for too long.
3. When preparing the chromogenic reagent, first transfer the powder into a glass container, then add a small amount of concentrated sulfuric acid, stir carefully to dissolve, then add the remaining concentrated sulfuric acid. The prepared chromogenic reagent can be stored at 4 °C, but for no more than 3 days. It is recommended to prepare fresh immediately before use.
4. The glucose standard is provided sterile. Once opened, storage at 4 °C may lead to contamination. For first use, open it in a clean bench and dispense aseptically before storing at 4 °C. If sterility cannot be guaranteed, aliquot and freeze at –20 °C.
5. This product is for research use only and is not suitable for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves when handling.
ZJ26F0636434
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