Difference between revisions of "Glycogen Determination from Tissues"

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m (Added step 2,clarified how to mix sample, added clarification to step 3 as to ensure all sample is immersed in KOH, added step 6, clarified the washing steps in order, clarified how to dry pellet)
 
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==Protocol==
 
==Protocol==
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# 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.  
+
# Weigh 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.  
 
# Turn on the heating block and set it to 95C (this can take up to 15 minutes to reach the desired temperature).
 
# Turn on the heating block and set it to 95C (this can take up to 15 minutes to reach the desired temperature).
 
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing by gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
 
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing by gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
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# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
 
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
 
# Quantify glucose using kit:
 
# Quantify glucose using kit:
−
## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
+
## Add 100 uL of Glucose buffer solution to each well, including wells for standard curve and blank
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## 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)
+
## Add 1-5 ul of glucose standard 200mg/dL diluted 1:5
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## Add 10 uL digested glycogen (for mice fasted more than 6 hours. for fed/short fast need less)
+
## Add 10 uL digested glycogen for fasted samples, and use a 10X dilution for refed tissues (using either 5 or 2ul from the diluted sample)  
 
## Mix and incubate at 37C for 5 min
 
## Mix and incubate at 37C for 5 min
 
## Measure absorbance at 505 nm
 
## Measure absorbance at 505 nm

Latest revision as of 13:02, 19 June 2017


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. (Sigma A7420-5MG)
  • 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. Weigh 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. Turn on the heating block and set it to 95C (this can take up to 15 minutes to reach the desired temperature).
  3. Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing by gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
  4. Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
  5. Boil for 5 min.
  6. Turn on the incubator and set it to 37C.
  7. Centrifuge at 13 000 RPM for 5 min.
  8. Aspirate the solution leaving the pellet at the bottom of the vial.
  9. Resuspend pellet in 200 uL water while making sure that all the glycogen dissolves in water, then add 400 uL ethanol.
  10. Boil 5 min, spin 5 min and Repeat wash steps twice more (Wash Steps: Aspirate -> Resuspend with H20 and EtOH -> Boil -> Centrifuge -> Aspirate).
  11. Dry pellet on the bench by leaving the vial cap open.
  12. 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
  13. Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
  14. Quantify glucose using kit:
    1. Add 100 uL of Glucose buffer solution to each well, including wells for standard curve and blank
    2. Add 1-5 ul of glucose standard 200mg/dL diluted 1:5
    3. Add 10 uL digested glycogen for fasted samples, and use a 10X dilution for refed tissues (using either 5 or 2ul from the diluted sample)
    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

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