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==Materials==
* Glycogen Synthase Buffer ('''GSB''')
** 50 mM HEPES
** 100 mM Sodium fluoride
** 10 mM EDTA
* Glycogen (Sigma catalog # ...)
* Glucose-6-Phosphate (Sigma catalog # G-7879). Dissolve to 200 mM in water
* UDP-Glucose (Sigma catalog # U-4625). Dissolve to 200 mM in water.
* 14C-UDP-Glucose (ARC # ARC 0154-50 µCi)
==Assay==
# Serum and glucose deprive cells for 3h in DMEM Low Glucose + 0.5% FBS.
# Treat cells as required
# Add GSB to cells (1 mL/10cm dish) and scrape
# Sonicate 10s then spin 5 min at 3500K in eppendorf centrifuge. Snap freeze supernatants if required, this decreases the absolute but not relative activity.
# Prepare reaction mixture (enough for 20 assays):
## 1 mL GSB
## 16 mg Glycogen (combine these two ahead of time and dissolve at 42C for 10-15 min)
## 60 uL of 200 mM UDP Glucose
## 1 uCi of 14C-UDP-Glucose
## 62.5 uL Water '''or''' 200 mM G6P
# Assay 50 uL of extract + 50 uL of reaction mixture. Do in triplicate -/+ G6P.
# Incubate 15 min at 37C with occasional shaking
# Place on ice for 15 min.
# Spot 90 uL of reaction mixture onto GF/A filters. Wait 3s and immerse in 70% ethanol at 4C
# Wash 15 min at 4C
# Wash twice 10-20 min at room temperature with fresh 70% ethanol.
# Air dry overnight and count in 5 mL scintillation fluid.
* Glycogen Synthase Buffer ('''GSB''')
** 50 mM HEPES
** 100 mM Sodium fluoride
** 10 mM EDTA
* Glycogen (Sigma catalog # ...)
* Glucose-6-Phosphate (Sigma catalog # G-7879). Dissolve to 200 mM in water
* UDP-Glucose (Sigma catalog # U-4625). Dissolve to 200 mM in water.
* 14C-UDP-Glucose (ARC # ARC 0154-50 µCi)
==Assay==
# Serum and glucose deprive cells for 3h in DMEM Low Glucose + 0.5% FBS.
# Treat cells as required
# Add GSB to cells (1 mL/10cm dish) and scrape
# Sonicate 10s then spin 5 min at 3500K in eppendorf centrifuge. Snap freeze supernatants if required, this decreases the absolute but not relative activity.
# Prepare reaction mixture (enough for 20 assays):
## 1 mL GSB
## 16 mg Glycogen (combine these two ahead of time and dissolve at 42C for 10-15 min)
## 60 uL of 200 mM UDP Glucose
## 1 uCi of 14C-UDP-Glucose
## 62.5 uL Water '''or''' 200 mM G6P
# Assay 50 uL of extract + 50 uL of reaction mixture. Do in triplicate -/+ G6P.
# Incubate 15 min at 37C with occasional shaking
# Place on ice for 15 min.
# Spot 90 uL of reaction mixture onto GF/A filters. Wait 3s and immerse in 70% ethanol at 4C
# Wash 15 min at 4C
# Wash twice 10-20 min at room temperature with fresh 70% ethanol.
# Air dry overnight and count in 5 mL scintillation fluid.