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.
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 20 mL of concentrated sulfuric acid (provided by the user), dissolve thoroughly, and store protected from light. This solution is valid for 3 days at 4 °C; prepare it and use as soon as possible.
3) Before use, dilute the 1 mg/mL glucose standard with double distilled water at a ratio of 1:99 to obtain a 0.01 mg/mL glucose working solution. This solution is valid only on the day of preparation. Note: 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..
2. 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.
3. Sample Detection
According to the table below, add the samples as indicated. After adding the chromogenic reagent, mix thoroughly before placing into the boiling water bath; otherwise, flocculent precipitates may appear. After mixing, boil in water for 5 min, then remove, cool, and mix again. Use a 1 cm pathlength cuvette, set zero with the blank tube at 620 nm, and measure the OD value of each tube.
Reagent | Blank tube | Standard tube | Sample tube |
Double distilled water (mL) | 1.0 | | |
0.01 mg/mL glucose (mL) | | 1.0 | |
Glycogen test solution (mL) | | | 1.0 |
Chromogenic reagent (mL) | 2.0 | 2.0 | 2.0 |
4. Result Calculation
1) Calculated by sample mass:
Glycogen content (mg/g) = Aₛₐₘₚₗₑ÷Aₛₜₐₙdₐᵣd× Mₛₜₐₙdₐᵣd × N ÷ 1.11
Mₛₜₐₙdₐᵣd : sugar content in the standard tube, 0.01 mg;
N: dilution factor of the sample before testing;
1.11: constant for converting glucose content measured by this method to glycogen content, i.e., the color intensity of 100 μg glycogen with the anthrone reagent is equivalent to that of 111 μg glucose with the anthrone reagent.
2) Calculated by sample protein concentration:
Glycogen content (mg/mg prot) = Aₛₐₘₚₗₑ ÷ Aₛₜₐₙdₐᵣd × C ₛₜₐₙdₐᵣd × N ÷1.11÷Cpr
C ₛₜₐₙdₐᵣd : standard concentration, 0.01 mg/mL;
Cpr: protein concentration of the cell homogenate, mg prot/mL;
1.11: constant for converting glucose content measured by this method to glycogen content, i.e., the color intensity of 100 μg glycogen with the anthrone reagent is equivalent to that of 111 μg glucose with the anthrone reagent.
5. 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. G1515911A is corrosive. Please handle with care. For your safety and health, please wear a lab coat and disposable gloves when handling.
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