Difference between revisions of "Glycogen Determination from Cells"

Revision as of 18:21, 5 October 2010

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).
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 glycogen. 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