Glycogen Determination from Cells - Revision history

Davebrid: clarified source of glycogen

2022-06-30T18:23:31Z

clarified source of glycogen

← Older revision		Revision as of 18:23, 30 June 2022
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	# Quantify glucose using kit:		# Quantify glucose using kit:
	## Add 1-5 uL glucose standard for standard curve		## 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.		## 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.
	## Mix and incubate at 37C for 5 min		## Mix and incubate at 37C for 5 min
	## Measure absorbance at 505 nm		## Measure absorbance at 505 nm

Davebrid: added cell lysis as per alan cheng

2010-10-06T13:17:00Z

added cell lysis as per alan cheng

← Older revision		Revision as of 13:17, 6 October 2010
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	==Protocol==		==Protocol==
	# Treat cells as desired and wash 2x with ice cold PBS.		# Treat cells as desired and wash 2x with ice cold PBS.
	# Scrape cells into 0.5 mL of Sodium Acetate (for 12 well).		# Scrape cells into 0.5 mL of Sodium Acetate (for 12 well). Lyse by either brief sonication (~10s) or 3x freeze thaw cycles.
	# Measure protein content by bradford assay for normalization		# Measure protein content by bradford assay for normalization
	# Quantify glucose using kit:		# Quantify glucose using kit:

Davebrid: wrote initial page

2010-10-05T18:21:33Z

wrote initial page

New page

==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]]