Difference between revisions of "Glycogen Determination from Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Wrote initial protocol) |
Davebridges (Talk | contribs) (added glucose quantification protocol) |
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* 50 mM Sodium Acetate, pH 4.8 | * 50 mM Sodium Acetate, pH 4.8 | ||
* Amyloglucosidease | * Amyloglucosidease | ||
| + | * Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091) | ||
| + | * Glucose standard solution (500 mg/dL; Wako) | ||
==Protocol== | ==Protocol== | ||
| Line 17: | Line 19: | ||
# Resuspend pellet in 200 uL of 50 mM Sodium Acetate, pH 4.8 | # Resuspend pellet in 200 uL of 50 mM Sodium Acetate, pH 4.8 | ||
# Add 0.3 mg/mL amyloglucosidase and place at 37C for 3h | # Add 0.3 mg/mL amyloglucosidase and place at 37C for 3h | ||
| − | # Quantify glucose | + | # Quantify glucose using kit: |
| + | ## Add 1-5 uL glucose standard for standard curve | ||
| + | ## Add 10 uL digested glycogen | ||
| + | ## Mix and incubate at 37C for 5 min | ||
| + | ## Measure absorbance at 505 nm | ||
| + | ## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10) | ||
Reference: | Reference: | ||
PMID 15282316 | PMID 15282316 | ||
Revision as of 16:57, 6 May 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
- Weight out 30-90 mg tissue into screw cap vial and record weights.
- Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing.
- Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
- Boil for 5 min.
- Centrifuge at 13 000 RPM for 5 min.
- Resuspend pellet in 200 uL water then add 400 uL ethanol. Boil 5 min, spin 5 min and Repeat wash steps twice more.
- Dry pellet
- Resuspend pellet in 200 uL of 50 mM Sodium Acetate, pH 4.8
- Add 0.3 mg/mL amyloglucosidase and place at 37C for 3h
- Quantify glucose using kit:
- Add 1-5 uL glucose standard for standard curve
- Add 10 uL digested glycogen
- Mix and incubate at 37C for 5 min
- Measure absorbance at 505 nm
- Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)
Reference:
