Difference between revisions of "Glycogen Determination from Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (added categories) |
Davebridges (Talk | contribs) (updated details about assay) |
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* Ethanol | * Ethanol | ||
* 50 mM Sodium Acetate, pH 4.8 | * 50 mM Sodium Acetate, pH 4.8 | ||
| − | * | + | * Amyloglucosidase 0.3 mg/mL |
| − | * Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091) | + | * 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 (500 mg/dL; Wako) | * Glucose standard solution (500 mg/dL; Wako) | ||
==Protocol== | ==Protocol== | ||
| − | # Weight out 30-90 mg tissue into screw cap vial and record weights. | + | # Weight out 30-90 mg tissue into *screw cap vial* and record weights. Screw cap vials are really important or else the lids will pop off |
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing. | # 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. | # Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol. | ||
| Line 21: | Line 21: | ||
# Centrifuge at 13 000 RPM 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. | # 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 | + | # Dry pellet on the bench |
| − | # | + | # 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 |
| − | # | + | # Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N |
# Quantify glucose using kit: | # Quantify glucose using kit: | ||
| − | ## Add 1-5 uL glucose standard for standard curve | + | ## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. |
| + | ## Add 1-5 uL glucose standard (200mg/dL) for standard curve | ||
## Add 10 uL digested glycogen | ## Add 10 uL digested glycogen | ||
## Mix and incubate at 37C for 5 min | ## Mix and incubate at 37C for 5 min | ||
Revision as of 15:02, 24 June 2011
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
- 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 (500 mg/dL; Wako)
Protocol
- Weight out 30-90 mg tissue into *screw cap vial* and record weights. Screw cap vials are really important or else the lids will pop off
- 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 on the bench
- 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
- Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
- Quantify glucose using kit:
- Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette.
- Add 1-5 uL glucose standard (200mg/dL) 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:
