Difference between revisions of "Glycogen Synthase Assay"

Revision as of 19:49, 30 March 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

1. Serum and glucose deprive cells for 3h in DMEM Low Glucose + 0.5% FBS.
2. Treat cells as required
3. Add GSB to cells (1 mL/10cm dish) and scrape
4. Sonicate 10s then spin 5 min at 3500K in eppendorf centrifuge. Snap freeze supernatants if required, this decreases the absolute but not relative activity.
5. Prepare reaction mixture (enough for 20 assays):
   1. 1 mL GSB
   2. 16 mg Glycogen (combine these two ahead of time and dissolve at 42C for 10-15 min)
   3. 60 uL of 200 mM UDP Glucose
   4. 1 uCi of 14C-UDP-Glucose
   5. 62.5 uL Water or 200 mM G6P
6. Assay 50 uL of extract + 50 uL of reaction mixture. Do in triplicate -/+ G6P.
7. Incubate 15 min at 37C with occasional shaking
8. Place on ice for 15 min.
9. Spot 90 uL of reaction mixture onto GF/A filters. Wait 3s and immerse in 70% ethanol at 4C
10. Wash 15 min at 4C
11. Wash twice 10-20 min at room temperature with fresh 70% ethanol.
12. Air dry overnight and count in 5 mL scintillation fluid.