Glycogen Synthase Assay: Difference between revisions
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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 | ||