Difference between revisions of "Glycogen Phosphorylase Assay"

Latest revision as of 16:44, 19 September 2011

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

1. Cells are starved and treated as desired.
2. Wash 3x with ice cold PBS.
3. 200 uL of homogenization buffer is added per well of a 6 well dish.
4. Cells are scraped then centrifuged at 13k for 10min. Lysates are transfered to fresh tubes.
5. Prepare radioactive reaction mixture.
   1. Need 2 x 40 uL reaction mixture per sample.
   2. Add 25 uL of 200 mM G1P and 2 uCi of radioactive G1P to 1mL Reaction Mixture.
   3. Assays are done +/- 3 mM 5'-AMP. Prepare one reaction mixture tube with and with 45 uL/mL AMP and one with water.
6. Add 20 uL Lysate to 40 uL radioactive reaction mixture.
7. Place at 37C for 20 min to react.
8. Place samples on ice for 15 min.
9. Spot 50 uL of mixture onto GF/A filter and let air dry for a few seconds. Place in a 6 well dish.
10. Add ice cold 70% Ethanol and washed 15-20 min at 4C.
11. Wash twice more with room temperature 70% ethanol for 15-20 min.
12. Air dry and count filters.