Glycogen Determination from Tissues: Difference between revisions

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* Ethanol

* 50 mM Sodium Acetate, pH 4.8

* ~~Amyloglucosidease~~

* 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)

==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.

# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.

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# 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

# Dry pellet on the bench

# ~~Resuspend pellet in 200 uL of~~ 50 mM Sodium Acetate, pH 4.8

# 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~~ amyloglucosidase and ~~place~~ at 37C for 3h

# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N

# 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

## Mix and incubate at 37C for 5 min

@@ Line 10: / Line 10: @@
 * Ethanol
 * 50 mM Sodium Acetate, pH 4.8
-* Amyloglucosidease
+* 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)
 ==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.
 # Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
@@ Line 21: / Line 21: @@
 # 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
+# Dry pellet on the bench
-# Resuspend pellet in 200 uL of 50 mM Sodium Acetate, pH 4.8
+# 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 amyloglucosidase and place at 37C for 3h
+# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
 # 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
 ## Mix and incubate at 37C for 5 min