906
edits
Changes
updated details about assay
* Ethanol
* 50 mM Sodium Acetate, pH 4.8
* AmyloglucosideaseAmyloglucosidase 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.
# 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 pelleton the bench# Resuspend pellet in 200 uL of 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# Add 0.3 mg/mL Resuspend pellets in 200uL amyloglucosidase solution and place 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