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==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==
# Treat cells as desired and wash 2x with ice cold PBS.
# Scrape cells into 0.5 mL of Sodium Acetate (for 12 well).
# Measure protein content by bradford assay for normalization
# Quantify glucose using kit:
## Add 1-5 uL glucose standard for standard curve
## Add 10 uL glycogen. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate.
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)
# Add amyloglucosidase (Add 0.3 mg/mL) and place at 37C for 3h-O/N
# Re-measure glucose levels
# 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
[[Category:Glycogen]]
[[Category:Metabolism]]
[[Category:Tissue Culture]]
* 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==
# Treat cells as desired and wash 2x with ice cold PBS.
# Scrape cells into 0.5 mL of Sodium Acetate (for 12 well).
# Measure protein content by bradford assay for normalization
# Quantify glucose using kit:
## Add 1-5 uL glucose standard for standard curve
## Add 10 uL glycogen. If signal is too low increase the volume. Also make a blank with the same volume of Sodium acetate.
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)
# Add amyloglucosidase (Add 0.3 mg/mL) and place at 37C for 3h-O/N
# Re-measure glucose levels
# 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
[[Category:Glycogen]]
[[Category:Metabolism]]
[[Category:Tissue Culture]]
