Difference between revisions of "Glycogen Determination from Tissues"

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(Wrote initial protocol)
 
(added glucose quantification protocol)
Line 6: Line 6:
 
* 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

  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