915
edits
Changes
copied protocol from alan cheng
==Materials==
* Homogenization Buffer: 50 mM MES (pH 6.1), 50 mM KF, 60 mM b-mercaptoethanol, 10 mM EDTA, 0.5% Triton X-100 with protease inhibitors
* Ice cold PBS
* Reaction Mixture: 1 mL of 400 mM KF, 10 mM EDTA, 30 mg of glycogen. Warmed at 42C until glycogen is dissolved.
* 200 mM Glucose-1-Phosphate
* 14C Glucose-1-Phosphate
* 100 mM 5'-AMP
* GF/A filter circles
* Ice cold 70% ethanol
==Protocol==
# Cells are starved and treated as desired.
# Wash 3x with ice cold PBS.
# 200 uL of homogenization buffer is added per well of a 6 well dish.
# Cells are scraped then centrifuged at 13k for 10min. Lysates are transfered to fresh tubes.
# Prepare radioactive reaction mixture.
## Need 2 x 40 uL reaction mixture per sample.
## Add 25 uL of 200 mM G1P and 2 uCi of radioactive G1P to 1mL Reaction Mixture.
## Assays are done +/- 3 mM 5'-AMP. Prepare one reaction mixture tube with and with 45 uL/mL AMP and one with water.
# Add 20 uL Lysate to 40 uL radioactive reaction mixture.
# Place at 37C for 20 min to react.
# Place samples on ice for 15 min.
# Spot 50 uL of mixture onto GF/A filter and let air dry for a few seconds. Place in a 6 well dish.
# Add ice cold 70% Ethanol and washed 15-20 min at 4C.
# Wash twice more with room temperature 70% ethanol for 15-20 min.
# Air dry and count filters.
[[Category: Glycogen]]
[[Cagegory: Assays]]
[[Category: Cell Based Assays]]
[[Category: Radioactive Assays]]
[[Category: Metabolism]]
* Homogenization Buffer: 50 mM MES (pH 6.1), 50 mM KF, 60 mM b-mercaptoethanol, 10 mM EDTA, 0.5% Triton X-100 with protease inhibitors
* Ice cold PBS
* Reaction Mixture: 1 mL of 400 mM KF, 10 mM EDTA, 30 mg of glycogen. Warmed at 42C until glycogen is dissolved.
* 200 mM Glucose-1-Phosphate
* 14C Glucose-1-Phosphate
* 100 mM 5'-AMP
* GF/A filter circles
* Ice cold 70% ethanol
==Protocol==
# Cells are starved and treated as desired.
# Wash 3x with ice cold PBS.
# 200 uL of homogenization buffer is added per well of a 6 well dish.
# Cells are scraped then centrifuged at 13k for 10min. Lysates are transfered to fresh tubes.
# Prepare radioactive reaction mixture.
## Need 2 x 40 uL reaction mixture per sample.
## Add 25 uL of 200 mM G1P and 2 uCi of radioactive G1P to 1mL Reaction Mixture.
## Assays are done +/- 3 mM 5'-AMP. Prepare one reaction mixture tube with and with 45 uL/mL AMP and one with water.
# Add 20 uL Lysate to 40 uL radioactive reaction mixture.
# Place at 37C for 20 min to react.
# Place samples on ice for 15 min.
# Spot 50 uL of mixture onto GF/A filter and let air dry for a few seconds. Place in a 6 well dish.
# Add ice cold 70% Ethanol and washed 15-20 min at 4C.
# Wash twice more with room temperature 70% ethanol for 15-20 min.
# Air dry and count filters.
[[Category: Glycogen]]
[[Cagegory: Assays]]
[[Category: Cell Based Assays]]
[[Category: Radioactive Assays]]
[[Category: Metabolism]]