Preparation of Protein Lysates from Mouse Tissues - Revision history

Reddj: added different RIPA volume for WAT

2019-04-25T16:06:00Z

added different RIPA volume for WAT

← Older revision		Revision as of 16:06, 25 April 2019
Line 7:		Line 7:
	#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.		#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.
	#Cool the centrifuge to 4C.		#Cool the centrifuge to 4C.
	#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL)		#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) for liver and muscle. Add 2-3uL/mg of RIPA for WAT.
	#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).		#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
	#Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.		#Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.

Reddj at 17:06, 9 April 2018

2018-04-09T17:06:28Z

← Older revision		Revision as of 17:06, 9 April 2018
Line 9:		Line 9:
	#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL)		#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL)
	#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).		#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
	#Remove stainless steel bead or transfer the homogenized tissue to a new tube.		#Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
	#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])		#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])
	#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer		#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
	#Heat samples with loading buffer at ~~95C~~ for 5 mins		#Heat samples with loading buffer at 85C for 2 mins
	#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80		#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80

Reddj at 20:20, 15 March 2018

2018-03-15T20:20:01Z

← Older revision		Revision as of 20:20, 15 March 2018
Line 11:		Line 11:
	#Remove stainless steel bead or transfer the homogenized tissue to a new tube.		#Remove stainless steel bead or transfer the homogenized tissue to a new tube.
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
	#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])		#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])
	#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer		#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer

Reddj: /* Protocol */

2017-11-02T20:10:35Z

Protocol

← Older revision		Revision as of 20:10, 2 November 2017
Line 6:		Line 6:
	#Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.		#Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
	#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.		#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.
	#Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)		#Cool the centrifuge to 4C.
			#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL)
	#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).		#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
			#Remove stainless steel bead or transfer the homogenized tissue to a new tube.
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify

Mpeloqu1: /* Protocol */

2013-07-26T21:27:00Z

Protocol

← Older revision		Revision as of 21:27, 26 July 2013
Line 10:		Line 10:
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
	#~~Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration~~ (see [[Bradford Assay]])		#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])
	#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer		#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
			#Heat samples with loading buffer at 95C for 5 mins
	#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80		#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80

Davebrid: updated protocol with new weights

2013-07-15T16:11:41Z

updated protocol with new weights

← Older revision		Revision as of 16:11, 15 July 2013
Line 1:		Line 1:
	==Materials==		==Materials==
	*RIPA Buffer (see [[Buffer/RIPA\|RIPA]])		*RIPA Buffer (see [[Buffer/RIPA\|RIPA]]) or other Lysis buffer. Add protease inhibitors.
	*Mouse Tissues		*Mouse Tissues (Frozen)

	==Protocol==		==Protocol==
	#Weigh frozen tissue samples, only need ~~100~~-~~300~~ mg of tissue. If there is too much cut it off and return the extra tissue to the -80		#Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
	~~#For fibrous tissues chop into small pieces with scissors~~		#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.
	#Add ~~3 volumes~~ of RIPA to tissue ~~and homogenize 20x with 1 mL glass homogenizer~~ (~~normally on SHC bench~~)		#Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)
	#~~Transfer lysate to fresh tube~~ and keep on ice		#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
	~~#Clean homogenizer~~ and ~~lyse remaining tissues~~
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
	#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see [[Bradford Assay]])		#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see [[Bradford Assay]])
	#~~Make 100~~ uL of ~~SDS sample using~~ 2X loading buffer		#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
	#Snap freeze remaining clarified lysate and store at -80		#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80

Davebrid: changed category

2012-05-28T14:42:07Z

changed category

← Older revision		Revision as of 14:42, 28 May 2012
Line 18:		Line 18:
	[[Category:Protein]]		[[Category:Protein]]
	[[Category:SDS-PAGE]]		[[Category:SDS-PAGE]]
	[[Category:Mouse]]		[[Category:Mouse Work]]
	[[Category:Tissues]]		[[Category:Tissues]]

Davebrid: added categories

2010-10-13T18:46:09Z

added categories

← Older revision		Revision as of 18:46, 13 October 2010
Line 14:		Line 14:
	#Make 100 uL of SDS sample using 2X loading buffer		#Make 100 uL of SDS sample using 2X loading buffer
	#Snap freeze remaining clarified lysate and store at -80		#Snap freeze remaining clarified lysate and store at -80


			[[Category:Protein]]
			[[Category:SDS-PAGE]]
			[[Category:Mouse]]
			[[Category:Tissues]]

Davebrid at 13:08, 8 July 2009

2009-07-08T13:08:10Z

← Older revision		Revision as of 13:08, 8 July 2009
Line 11:		Line 11:
	#Centrifuge at 14 000 RPM at 4C for 10 min		#Centrifuge at 14 000 RPM at 4C for 10 min
	#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
	#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration.		#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see [[Bradford Assay]])
	#Make 100 uL of SDS sample using 2X loading buffer		#Make 100 uL of SDS sample using 2X loading buffer
	#Snap freeze remaining clarified lysate and store at -80		#Snap freeze remaining clarified lysate and store at -80

Davebrid: wrote initial page

2009-07-08T13:07:29Z

wrote initial page

New page

==Materials==
*RIPA Buffer (see [[Buffer/RIPA|RIPA]])
*Mouse Tissues

==Protocol==
#Weigh frozen tissue samples, only need 100-300 mg of tissue. If there is too much cut it off and return the extra tissue to the -80
#For fibrous tissues chop into small pieces with scissors
#Add 3 volumes of RIPA to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench)
#Transfer lysate to fresh tube and keep on ice
#Clean homogenizer and lyse remaining tissues
#Centrifuge at 14 000 RPM at 4C for 10 min
#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration.
#Make 100 uL of SDS sample using 2X loading buffer
#Snap freeze remaining clarified lysate and store at -80