Digestion for Glycogen determination in liver/muscle

[from Allen Saltiel's lab]

Digestion for glycogen determination:

• 1. Place muscles (80-100 mg)/liver 50-60 mg in 500 µL 30% KOH

  2. Heat at 100 C for 10 minutes (or until dissolved)

  3. After cool down, add 100 µL 20% Na₂SO₄ to digest, vortex

  4. Add 1.0 mL EtOH, vortex, and then parafilm tubes. Precipitate overnight at –20 C

  5. Centrifuge at 3000 xg for 10 min. Discard Sup

  6. Wash pellet with 2 mL 70% EtOH, vortex thoroughly, then centrifuge at 3000 xg for 0 min (break up pellet with vortex after 1 mL)

  7. Discard sup, and resuspend pellet in 4 N H₂SO₄ (250 µL for muscle, 500 µL for liver), heat for 100 C for 10 minutes

  8. Neutralize with equal volume 4N NaOH

Glycogen assay:

• • Muscle: when starting muscle weight is between 80-100 mg precipitate 200 µL neutralized extract with BaOH/ZnSO₄ (0/9 mL each). Shake by inverting; spin 1-2 min at 3000 xg. Use 150 µL aliquots of supernatant for glucose assay when using 150 µL of Glucose- INF (340 nm).

  • Liver: When starting liver weight is between 50-60 mg (and final pellet is resuspended in 1 mL H₂SO₄ + NaOH), precipitate 200 µL aliquot neutralized extract with BaOH/ZnSO4 (0.9 mL each). Use 150 µL aliquots of supernatant for glucose assay when using 150 µL of Glucose-INF (340 nm).

Reagents:

• • Glucose-INF (stock#: 17-2100P)

  • Barium hydroxide solution, 0.3 N (sigma 14-3)

  • Zinc sulfate solution, 0.3 N (sigma 14-4)

  • 30% KOH: 87.58 g KOH in 292 mL ddH₂O

  • 20% Na2SO4: 40 g Na₂SO₄ in 200 mL ddH₂O

  • 4N H2SO4: 11.1 mL 36 N H2SO4 in 100 mL ddH₂O

  • 4N NaOH: 19.43 g NaOH in 121.4 mL ddH₂O

  • Glucose standard (STD)

Weight 190.5 mg of D-(+)-Glucose (FW=180.2, stock # G8270) into a 10.57 ml ddH₂O (=100 µmol/mL), then dilute into 10 µmol/mL, 1 µmol/mL, 0.5 µmol/mL, 0.1 µmol/mL, 0.05 µmol/mL, 0.025 µmol/mL, 0.01 µmol/mL.

Glycogen type IV (sigma G0885) –8.0 mg in 8 mL H₂O (take 100 µL for assay)