Glycogen Determination from Cells

Materials and Buffers

• 50 mM Sodium Acetate, pH 4.8
• Amyloglucosidease
• Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091)
• Glucose standard solution (500 mg/dL; Wako)

Protocol

1. Treat cells as desired and wash 2x with ice cold PBS.
2. Scrape cells into 0.5 mL of Sodium Acetate (for 12 well). Lyse by either brief sonication (~10s) or 3x freeze thaw cycles.
3. Measure protein content by bradford assay for normalization
4. Quantify glucose using kit:
   1. Add 1-5 uL glucose standard for standard curve
   2. Add 10 uL of the glycogen containing sample. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate.
   3. Mix and incubate at 37C for 5 min
   4. Measure absorbance at 505 nm
   5. Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)
5. Add amyloglucosidase (Add 0.3 mg/mL) and place at 37C for 3h-O/N
6. Re-measure glucose levels
7. Glycogen levels are calculated by the difference in glucose levels before and after glycosidase treatment and are presented in equivalent glucose units/mg protein.

Reference:

PMID 15282316