Difference between revisions of "Glycogen Synthase Assay"
From Bridges Lab Protocols
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# Incubate 15 min at 37C with occasional shaking | # Incubate 15 min at 37C with occasional shaking | ||
# Place on ice for 15 min. | # Place on ice for 15 min. | ||
| − | # Spot 90 uL of reaction mixture onto GF/A filters. | + | # Spot 90 uL of reaction mixture onto GF/A filters. |
| + | # Place in a well of a 6 well dish. | ||
| + | # Add ~2mL of ice cold 70% ethanol. | ||
# Wash 15 min at 4C | # Wash 15 min at 4C | ||
# Wash twice 10-20 min at room temperature with fresh 70% ethanol. | # Wash twice 10-20 min at room temperature with fresh 70% ethanol. | ||
| − | # Air dry overnight and count in 5 mL scintillation fluid. | + | # Air dry overnight on a paper towel and count in 5 mL scintillation fluid. Also count 5 uL of both the - and + G6P solution to calculate total incorporation |
Revision as of 15:24, 27 April 2010
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.
- Place in a well of a 6 well dish.
- Add ~2mL of ice cold 70% ethanol.
- Wash 15 min at 4C
- Wash twice 10-20 min at room temperature with fresh 70% ethanol.
- Air dry overnight on a paper towel and count in 5 mL scintillation fluid. Also count 5 uL of both the - and + G6P solution to calculate total incorporation
