Difference between revisions of "Glycogen Determination from Tissues"

Revision as of 18:21, 5 October 2010

Materials and Buffers

• Screw Capped Vials
• 30% KOH, prepared fresh
• 1M Sodium Sulfate
• Ethanol
• 50 mM Sodium Acetate, pH 4.8
• Amyloglucosidease
• Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091)
• Glucose standard solution (500 mg/dL; Wako)

Protocol

1. Weight out 30-90 mg tissue into screw cap vial and record weights.
2. Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing.
3. Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
4. Boil for 5 min.
5. Centrifuge at 13 000 RPM for 5 min.
6. Resuspend pellet in 200 uL water then add 400 uL ethanol. Boil 5 min, spin 5 min and Repeat wash steps twice more.
7. Dry pellet
8. Resuspend pellet in 200 uL of 50 mM Sodium Acetate, pH 4.8
9. Add 0.3 mg/mL amyloglucosidase and place at 37C for 3h
10. Quantify glucose using kit:
    1. Add 1-5 uL glucose standard for standard curve
    2. Add 10 uL digested glycogen
    3. Mix and incubate at 37C for 5 min
    4. Measure absorbance at 505 nm
    5. Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

Reference:

PMID 15282316