Glycogen Determination from Tissues - Revision history

Elhabbal at 13:02, 19 June 2017

2017-06-19T13:02:29Z

← Older revision		Revision as of 13:02, 19 June 2017
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	==Protocol==		==Protocol==
	# ~~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.

Mollyec: /* Protocol */

2017-06-15T16:55:16Z

Protocol

← Older revision		Revision as of 16:55, 15 June 2017
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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
	## 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
	## 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

Elhabbal: 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

2017-06-14T20:58:28Z

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

← Older revision		Revision as of 20:58, 14 June 2017
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	==Protocol==		==Protocol==
	# 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		# 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.
	# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing.		# 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.
	# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.		# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
	# Boil for 5 min.		# Boil for 5 min.
			# Turn on the incubator and set it to 37C.
	# 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.		# Aspirate the solution leaving the pellet at the bottom of the vial.
	# Dry pellet on the bench		# Resuspend pellet in 200 uL water while making sure that all the glycogen dissolves in water, then add 400 uL ethanol.
			# Boil 5 min, spin 5 min and Repeat wash steps twice more (Wash Steps: Aspirate -> Resuspend with H20 and EtOH -> Boil -> Centrifuge -> Aspirate).
			# Dry pellet on the bench by leaving the vial cap open.
	# 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		# 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		# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N

Davebrid: updated product number for amyloglucosidase

2014-06-24T12:50:23Z

updated product number for amyloglucosidase

← Older revision		Revision as of 12:50, 24 June 2014
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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 in 50 mM Sodium Acetate. Stored in -80		* 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 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)		* Glucose standard solution (200 or 500 mg/dL; Wako)

Irith at 14:26, 8 June 2012

2012-06-08T14:26:55Z

← Older revision		Revision as of 14:26, 8 June 2012
Line 27:		Line 27:
	## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)		## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
	## 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 glucose standard (500mg/dL) for standard curve (for microplate add 1-5 ul of glucose standard 200mg/dL diluted 1:5)
	## Add 10 uL digested glycogen		## Add 10 uL digested glycogen (for mice fasted more than 6 hours. for fed/short fast need less)
	## 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

Irith at 14:03, 8 June 2012

2012-06-08T14:03:08Z

← Older revision		Revision as of 14:03, 8 June 2012
Line 25:		Line 25:
	# 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.		## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
	## Add 1-5 uL glucose standard (500mg/dL) for standard curve		## 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 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

Davebrid: changed category

2012-05-28T15:07:04Z

changed category

← Older revision		Revision as of 15:07, 28 May 2012
Line 2:		Line 2:
	[[Category:Metabolism]]		[[Category:Metabolism]]
	[[Category:Mouse Tissues]]		[[Category:Mouse Tissues]]
	[[Category:~~Mice~~]]		[[Category:Mouse Work]]

	==Materials and Buffers==		==Materials and Buffers==

Davebrid: added sweave template

2012-05-21T14:40:47Z

added sweave template

← Older revision		Revision as of 14:40, 21 May 2012
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	## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)		## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

			==Calculations==
			# Use this Sweave template: https://raw.github.com/davebridges/biomolecule-scripts/master/R/Sweave/glycogen-analysis-tissue.Rnw
	Reference:		Reference:

	PMID 15282316		PMID 15282316

Davebrid: bolded vial type

2012-05-18T18:21:20Z

bolded vial type

← Older revision		Revision as of 18:21, 18 May 2012
Line 15:		Line 15:

	==Protocol==		==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		# 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
	# 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.

Davebrid: described preparation and storage of amyloglucosidase

2012-01-13T16:42:41Z

described preparation and storage of amyloglucosidase

← Older revision		Revision as of 16:42, 13 January 2012
Line 10:		Line 10:
	* Ethanol		* Ethanol
	* 50 mM Sodium Acetate, pH 4.8		* 50 mM Sodium Acetate, pH 4.8
	* Amyloglucosidase 0.3 mg/mL		* Amyloglucosidase 0.3 mg/mL in 50 mM Sodium Acetate. Stored in -80
	* Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091; for protocol see http://www.wakodiagnostics.com/pi/pi_autokit_glucose.pdf)		* 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)		* Glucose standard solution (200 or 500 mg/dL; Wako)