Glycogen Assay Kit (Colorimetric)

Catalog No. ABIN5067565

Product Details

Reactivity: Fusarium

Detection Method: Colorimetric

Application: Biochemical Assay (BCA)

Purpose: Glycogen Assay Kit measures total glycogen within biological samples.

Sample Type: Urine, Plasma, Serum

Characteristics: Glycogen Assay Kit is a simple colorimetric assay that measures the amount of total glycogen present in biological samples in a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays*, including blanks, glycogen standards, and unknown samples. Sample glycogen concentrations are determined by comparison with a known glycogen standard. The kit has a detection sensitivity limit of 3.8 μM glycogen. *Note: Each sample replicate requires 2 assays, one treated with amyloglucosidase (+AG) and one without (-AG). Glycogen is calculated from the difference in OD readings from the 2 wells.

Components:

1. Glycogen Standard : One 50 μL tube at 3 mM.
2. 10X Assay Buffer : One 25 mL bottle of 500 mM sodium phosphate pH 7.4.
3. Colorimetric Probe : One 50 μL tube in DMSO.
4. HRP : One 10 μL tube of a 100 U/mL solution in glycerol.
5. Amyloglucosidase : One 1 mL tube at 15 U/mL. Note: One unit is defined as the amount of enzyme that will release 1.0 micromole of glucose per minute at pH 4.8 at 60°C. 3
6. Glucose Oxidase : One 100 μL tube at 200U/mL. Note: One unit is defined as the amount of enzyme that will oxidize 1.0 micromole of beta-D- glucose to D-gluconic acid and hydrogen peroxide per minute at pH 5.1 at 35°C.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Suitable for use with serum, plasma, urine, lysates, and cell culture supernatants
• Detection sensitivity of 0.12uM
• Glycogen standard included

Protocol: Glycogen is broken down into glucose monomers by amyloglucosidase first, glucose is then oxidized by glucose oxidase into D-gluconic acid and hydrogen peroxide. The resulting hydrogen peroxide is then detected with a highly specific colorimetric probe. Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide, which bind in a 1:1 ratio. Samples are compared to a known concentration of glycogen standard within the 96-well microtiter plate format. Samples and standards are incubated for 45 minutes and then read with a standard 96-well colorimetric plate reader .

Reagent Preparation:

• 1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution. Stir or vortex to homogeneity. Store at room temperature.
• Reaction Mix: Prepare a Reaction Mix by diluting the Colorimetric Probe 1:100, HRP 1:500, and Glucose Oxidase 1:50 in 1X Assay Buffer. For example, add 10 μL Colorimetric Probe stock solution, 2 μL HRP stock solution, and 20 μL of Glucose Oxidase to 968 μL of 1X Assay Buffer for a total of 1 mL. This Reaction Mix volume is enough for 20 assays. The Reaction Mix is stable for 1 day at 4 °C. Note: Prepare only enough for immediate use by scaling the above example proportionally. 4

Sample Preparation:

• Cell culture supernatants: Cell culture media containing glucose should be avoided. To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The cell conditioned media may be assayed directly or diluted as necessary in PBS. Note: Maintain pH between 7and 8 for optimal working conditions as the Colorimetric Probe is unstable at high pH (>8.5).
• Tissue lysates: Sonicate or homogenize tissue sample in PBS and centrifuge at 10000 x g for 10 minutes at 4 °C. The supernatant may be assayed directly or diluted as necessary in PBS.
• Cell lysates: Resuspend cells at 1-2 x 106 cells/mL in PBS. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates may be assayed undiluted or diluted as necessary in PBS.
• Serum, plasma or urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant may be assayed directly or diluted as necessary in PBS. Notes:
• All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the Colorimetric Probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL (Votyakova and Reynolds, Ref. 2).
• Avoid samples containing DTT or β-mercaptoethanol since the Colorimetric Probe is not stable in the presence of thiols (above 10 μM).

Assay Procedure:

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate. Note: Each sample replicate requires two paired wells, one to be treated with Amyloglucosidase and one without the enzyme to measure endogenous glucose background (PBS will be added in place of Amyloglucosidase).
2. Add 50 μL of each glycogen standard or unknown sample into wells of a 96-well microtiter plate.
3. Add 10 μL of Amyloglucosidase to the standards and to one half of the paired sample wells and mix the well contents thoroughly.
4. Add 10 μL of PBS to the other half of the paired sample wells and mix thoroughly.
5. Incubate for 30 minutes at 37 °C.
6. Add 50 μL of Reaction Mix to each well. Mix the well contents thoroughly and incubate for 45 minutes at 37 °C protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the reaction kinetics.
7. Read the plate with a spectrophotometric microplate reader in the 540-570 nm range. Calculation of Results
   1. Determine the average absorbance values for each sample, control, and standard.
   2. Subtract the average zero standard value from itself and all standard values.
   3. Graph the standard curve (see Figure 2).
   4. Subtract the sample well values without Amyloglucosidase (-AG) from the sample well values containing enzyme (+AG) to obtain the difference. The absorbance difference is due to the Amyloglucosidase activity: ΔOD = (OD+AG) - (OD-AG)
   5. Compare the ΔOD of each sample to the standard curve to determine and extrapolate the quantity of glycogen present in the sample. Only use values within the range of the standard curve.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: RT/-20 °C

Storage Comment: Upon receipt, store the Glycogen Standard, Colorimetric Probe, HRP, Amyloglucosidase, and Glucose Oxidase at -20°C. The Colorimetric Probe is light sensitive and must be stored accordingly. Avoid multiple freeze/thaw cycles. Store the 10X Assay Buffer at room temperature. Note: After thawing Amyloglucosidase for the first time, make smaller aliquots and store at -20°C.

References

Pastore, Vainshtein, Klisch, Armani, Huynh, Herz, Polishchuk, Sandri, Ballabio: "TFE3 regulates whole-body energy metabolism in cooperation with TFEB." in: EMBO molecular medicine, Vol. 9, Issue 5, pp. 605-621, (2017) (PubMed).

Target Details

Background: Glycogen is a polysaccharide found in animals as well as simpler organisms such as fungi. Glycogen is made up of glucose monomers and is considered the primary method of storing glucose in animals. In humans, glycogen is mainly synthesized in the liver (up to 6 % of the total mass) and the muscles (approximately 2 % of the total mass). Lesser amounts of glycogen are found in the kidneys, glial cells in the brain, and white blood cells. Glycogen also serves as an energy source in the uterus during pregnancy to supply glucose to the fetus. While fats in adipose tissue represent the main stored energy source, glycogen is the second most abundant stored energy source. Glycogen is similar in structure and function to starch, a polysaccharide composed of glucose monomers that also serves as energy storage in plants. Glycogen differs structurally from starch in that it is more extensively branched and compact than starch. Glycogen storage diseases (GSD) are a set of disorders caused by the disruption of glycogen metabolism. Disruption of the liver's ability to supply the rest of the body with glycogen can lead to low blood sugar levels. Additionally, if liver glycogen is not broken down effectively, this can lead to abnormal enlargement of the liver. Glycogen synthase mutations causing impaired protein function causes lower glycogen synthesis and causes the liver to be small. Finally, glycogen brancher deficiency can cause aberrant forms of glycogen to be stored in the liver ultimately causing a disorder of progressive liver dysfunction.