Difference between revisions of "Glycogen Determination from Tissues"

Revision as of 14:26, 8 June 2012

Materials and Buffers

• Screw Capped Vials
• 30% KOH, prepared fresh
• 1M Sodium Sulfate
• Ethanol
• 50 mM Sodium Acetate, pH 4.8
• Amyloglucosidase 0.3 mg/mL in 50 mM Sodium Acetate. Stored in -80
• Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091; for protocol see http://www.wakodiagnostics.com/pi/pi_autokit_glucose.pdf)
• Glucose standard solution (200 or 500 mg/dL; Wako)

Protocol

1. Weight out 30-90 mg tissue into a screw cap vial and record weights. Screw cap vials are really important or else the lids will pop off
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 on the bench
8. Prepare amyloglucosidase solution by diluting the AG stock 100X into 50 mM Sodium Acetate, pH 4.8. Prepare enough for 200 uL per tube plus some extras
9. Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
10. Quantify glucose using kit:
    1. Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
    2. Add 1-5 uL glucose standard (500mg/dL) for standard curve (for microplate add 1-5 ul of glucose standard 200mg/dL diluted 1:5)
    3. Add 10 uL digested glycogen (for mice fasted more than 6 hours. for fed/short fast need less)
    4. Mix and incubate at 37C for 5 min
    5. Measure absorbance at 505 nm
    6. Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

Calculations

1. Use this Sweave template: https://raw.github.com/davebridges/biomolecule-scripts/master/R/Sweave/glycogen-analysis-tissue.Rnw

Reference:

PMID 15282316