UA-Glo® Glycogen Luminescence Detection Kit

The UA-Glo® Glycogen Luminescence Assay Kit detects glycogen content in samples through a two-step reaction. The first step involves the digestion of glycogen into glucose by glucoamylase (1,4-α-D-Glucan glucohydrolase). In the second step, the glucose content is measured via a coupled reaction involving glucose dehydrogenase, reductase, and luciferase. Glucose dehydrogenase acts on glucose to reduce NADP to NADPH, while reductase utilizes NADPH to reduce the reductase substrate (also a luciferin precursor) into luciferin. Finally, luciferin is catalyzed by luciferase to produce luminescence, the intensity of which is directly proportional to the glucose concentration.

The UA-Glo® Glycogen Luminescence Assay Kit is a homogeneous detection reagent. Its simple two-step procedure minimizes experimental errors caused by multiple pipetting steps. This kit offers high signal-to-noise ratio, excellent reproducibility, and stability, making it suitable for batch processing of various sample types.

The components and specifications of the UA-Glo® Glycogen Luminescence Assay Kit are as follows. The detectable 96-well plate reactions are calculated based on adding 25 μL of sample, 25 μL of glucoamylase digestion solution, and 50 μL of glucose detection reagent per well.

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The UA-Glo® Glycogen Assay Kit detects glycogen content through glucose amylase digestion of glycogen into glucose, followed by coupled reactions involving glucose dehydrogenase, reductase, and luciferase to measure glucose levels. The luciferase reaction offers extremely high sensitivity, but parameters such as pH, temperature, and experimental system setup significantly influence the results. Under most experimental conditions, direct comparison of absolute readings between samples is not recommended. It is advised to include the same reference compounds and solvent vehicle controls on each assay plate, with sample readings normalized to these controls before data processing and comparison. Alternatively, glycogen standards processed in the same manner as samples may be included on each plate to quantify glycogen content.

3.1 Sample Processing

The UA-Glo® Glycogen Assay Kit can be used to measure glycogen concentration in cell lysates and tissue homogenates. The linear detection range for glycogen extends up to 20 μg/mL. If sample glycogen concentration exceeds 20 μg/mL, refer to the table below for appropriate sample processing and dilution, or optimize dilution based on experimental requirements.

It is recommended to use the kit-supplied 0.3N HCl and Tris buffer (450mM Tris, pH 8.0) for sample processing. Acid treatment with 0.3N HCl rapidly lyses cells, halts metabolism, inactivates cellular enzymes, and degrades reduced NAD(P)H, preventing interference in subsequent glycogen detection. After acid treatment, adding Tris buffer slightly increases pH without negating the acid treatment effect. The combination of 0.3N HCl and Tris buffer (450mM Tris, pH 8.0) ensures the pH reaches the optimal range (~pH 5) for glucose amylase reaction after adding the digestion buffer. Samples treated with 0.3N HCl and Tris buffer can be immediately assayed or stored at -20°C or below for future use. For difficult-to-lyse samples (e.g., 3D cultures), 0.2% Triton X-100 may be added to 0.3N HCl to enhance metabolite release.

For tissue homogenization, use 1 volume of PBS mixed with 1/2 volume of 0.3N HCl, followed by addition of 1/2 volume of Tris buffer. Alternatively, homogenize tissue first, then sequentially add 0.3N HCl and Tris buffer. Tissues with high glycogen content (e.g., liver) require dilution to appropriate concentrations before assay, using a diluent of PBS + 0.3N HCl + Tris buffer in a 2:1:1 volume ratio.

3.2 Glycogen Assay Protocol

3.2.1 Sample Glycogen Detection

1)Add 25 μL of processed sample (from 3.1) per well in a 96-well white opaque-bottom plate. Negative controls should use glycogen/glucose-free medium or buffer processed identically to samples; positive controls may use glycogen standards similarly processed. Luciferase reactions are temperature-sensitive. Equilibrate reagents and samples to room temperature (22-25°C) and maintain stable temperature (±1°C) during assay.

2)Thaw glucose amylase buffer completely at room temperature, mix by inversion, and aliquot as needed. Remaining buffer may be stored at -20°C.

3)Thaw glucose amylase at room temperature and keep on ice. Prepare 1× digestion solution by diluting 200× stock with glucose amylase buffer, mixing thoroughly. Prepare fresh for each use; do not store. Aliquot remaining enzyme and store at -80°C.

4)Add 25 μL digestion solution per well, mix by plate shaking for 1 min.

5)Incubate at room temperature for 60 min.

6)Thaw luciferase detection reagent completely, mix by inversion, and equilibrate to room temperature. Aliquot remaining reagent and store at -80°C. Avoid >3 freeze-thaw cycles.

7)Prepare glucose detection reagent: Thaw reductase substrate at room temperature, keep on ice. Add to luciferase detection reagent at 200× dilution, mix thoroughly. Use immediately to avoid increased background. Prepare fresh; do not store. Aliquot remaining substrate and protect from light at -80°C (≤3 freeze-thaw cycles).

8)Add 50 μL detection reagent per well. Maintain a 1:1:2 ratio of sample:digestion reagent:detection reagent. For 384-well low-volume plates, use 5 μL sample + 5 μL digestion reagent + 10 μL detection reagent.

9)Mix by shaking for 1 min, then incubate in darkness for 60-90 min. Signal increases linearly until glucose is depleted.

10)Measure luminescence using a plate reader.

3.2.2 Glycogen Detection in Cultured Cell Lysates

1)Seed cells at appropriate density in 96-well plates (e.g., 5,000-50,000 cells/well).

2)Perform experimental treatments as required.

3)Aspirate medium and wash cells five times with 200 μL cold PBS to remove residual glucose. Perform washes rapidly to minimize glycogen metabolism changes.

4)Add 30 μL PBS per well.

5)Add 15 μL 0.3N HCl, mix by shaking for 5 min. Alternatively, premix PBS and 0.3N HCl (2:1) and add 45 μL/well.

6)Add 15 μL Tris buffer (450mM, pH 8.0), mix by shaking for 1 min.

7)Samples may be stored at ≤-20°C for future analysis.

8)Transfer 25 μL processed samples, controls to white opaque-bottom plates. For samples with high glucose (e.g., tissues), run duplicates: one well with digestion reagent, one with buffer alone. The signal difference represents glycogen content.

9)Proceed with glycogen detection as in 3.2.1.

1) Different batches are not recommended for mixed use

2) It is not recommended to change the amount of detection reagents without strict validation

3) For research use only