1. Sample preparation

• Homogenize 10⁶ (animal) cells with 200 µL dH₂O on ice.
  • approximately 10⁹ E.coli cells (1 ml culture with OD 1.25) ^{[9] [10]}
• Sonicate cells
• Boil homegenates for 10 minutes.
• Spin/centrifuge at 18 000 g for 10 minutes. The supernatant is now ready for assay!
• Add 2-50 µL samples to a 96-well pate.
• Adjust volume to 50 µL/well with Hydrolysis Buffer

Notes:

• For samples having glucose background, prepare well(s) containing same amont of sample as in the test well as background control.
• (Accurate determination of Glycogen in the test samples is best determined via spiking samples with a known amount of standard (example given 0.8 µg)

2. Standard Curve preparation

• Dilute the glycogen standard to 0.02 mg/mL by adding 10 µL of the Standard to 990 µL of distilled water, mix well.
• Add 0, 2, 4, 8, 10 µL to a series of wells.
• Adjust volume to 50 µL/well with Hydrolis Buffer to generate 0, 0.04, 0.08, 0.12, 0.16, and 0.2 µg per well

3. Add 1 µL Hydrolysis Enzyme mix to Standards and samples and mix well.

• for dilution samples, dilute with hydrolysis buffer

Incubate for 30 minutes at room temperature.

Note:

If glucose is present in your sample, you may do a glucose control without addition of hydrolysis enzyme to determine the level of glucose background.

4. Reaction Mix

• Mix enough reagents for the number of samples and standards to be performed
• For each well prepare a total 50 µL Reaction Mix.
  • Dev. Buffer: 48.7 µL
  • Dev. Enzyme Mix: 1 µL
  • OxiRed Probe: 0.3 µL
• Add 50 µL of the Reaction Mix to each well containing the Glycogen Standard or samples, mix well.

5. Measurement

• Incubate the reaction for 30 minutes at room temperature protected from light.
  • Fluorescence (Ex/Em = 535/587 nm)

6. Calculation

• Correct background by subtracting the 0 Glycogen Standard sample value (see 2.) from all readings.
• Apply sample readings to the standard curve to get B µg of glycogen.

Sample c_(Glycogen) = B / V * D µg/µL

B is the amount of Glycogen from Standard curve (µg)

V is the sample volume added into the reaction well (µL)

D is the sample dilution factor

• For spiked samples, correct for any sample interference by subtracting the sample reading from spiked sample.

For Spiked samples:

B = [OD_(sample corrected) / OD_(sample+glycogen Standard (corrected)) - OD_(sample corrected)] * Glycogen Spike (µg)

Notes about Glycogen:

• Glycogen molecular size: 60 000 molecules
  • Molecular Weight (MW) ~ 10⁶-10⁷ daltons
  • Glucose MW: 180.16 g/mol

Glycogen Assay Protocol short version

1. Sample preparation

• Homogenize 10⁶ cells with 200 µL dH₂O on ice.
• Boil homegenates for 10 minutes.
• Spin/centrifuge at 18 000 g for 10 minutes. The supernatant is now ready for assay!
• Add 2-50 µL samples to a 96-well pate.
• Adjust volume to 50 µL/well with Hydrolysis Buffer

2. Standard Curve preparation

• Dilute the glycogen standard to 0.02 mg/mL by adding 10 µL of the Standard to 990 µL of distilled water, mix well.
• Add 0, 2, 4, 8, 10 µL to a series of wells.
• Adjust volume to 50 µL/well with Hydrolis Buffer to generate 0, 0.04, 0.08, 0.12, 0.16, and 0.2 µg per well

3. Add 1 µL Hydrolysis Enzyme mix to Standards and samples and mix well.

• Incubate for 30 minutes at room temperature.

4. Reaction Mix

• Mix enough reagents for the number of samples and standards to be performed
• For each well prepare a total 50 µL Reaction Mix.
  • Dev. Buffer: 48.7 µL
  • Dev. Enzyme Mix: 1 µL
  • OxiRed Probe: 0.3 µL
• Add 50 µL of the Reaction Mix to each well containing the Glycogen Standard or samples, mix well.

5. Measurement

• Incubate the reaction for 30 minutes at room temperature protected from light.
  • Fluorescence (Ex/Em = 535/587 nm)

Pre-extraction of glycogen

• Another way to subtract the glucose background, is the pre-extraction of glycogen
• This procedure removes the majority of glucose background with minimal effect on glycogen

1. Take tissue or cells to a final content of 30-50 % in 30 % KOH
2. Heat to 100 °C for 2 h
3. Cool and add 2 volumes of 95% ethanol to precipitate crude glycogen
4. Centrifuge (16000 g for 10 min) and collect the precipitate
5. Suspend the pellet in a minimal amount of distilled water (25 ml original cell culture -> 5 ml were enough)
6. Acidify sample to pH 3 with 5 M HCl
7. Add 1 volume of ethanol to reprecipitate
8. Repeat steps 4-7 two more times, then let sample dry over night (pre-weigh container for drying)
9. The dried material can be weighed and dissolved for analysis