Preparation of Protein Lysates from Cells - Revision history

Iharvey at 21:43, 5 January 2018

2018-01-05T21:43:39Z

← Older revision		Revision as of 21:43, 5 January 2018
Line 11:		Line 11:
	#Remove supernatant to clean tube. If lysing fat cells, try to avoid the floating fat cake. If necessary respin to clarify		#Remove supernatant to clean tube. If lysing fat cells, try to avoid the floating fat cake. If necessary respin to clarify
	#Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.		#Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.
	#Heat samples with loading buffer at ~~95C~~ for 5 mins		#Heat samples with loading buffer at 85C for 2-3 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

Iharvey at 18:33, 15 December 2017

2017-12-15T18:33:26Z

← Older revision		Revision as of 18:33, 15 December 2017
Line 9:		Line 9:
	#Incubate on ice for 15 minutes		#Incubate on ice for 15 minutes
	#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 cells, try to avoid the floating fat cake. If necessary respin to clarify
	~~#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])~~		#Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.
	#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 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

Iharvey: Created page with "==Materials== RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells..."

2017-12-15T18:28:33Z

Created page with "==Materials== *RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. *Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells..."

New page

==Materials==
*RIPA Buffer (see [[Buffer/RIPA|RIPA]]) or other Lysis buffer. Add protease inhibitors.
*Cells (fresh or frozen)

==Protocol==
#Cool centrifuge to 4C
#If cells are not already frozen in buffer, add ~400ul RIPA plus PI while keeping cells on ice
#Scrape cells and transfer to cold micro centrifuge tube on ice
#Incubate on ice for 15 minutes
#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
#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
#Heat samples with loading buffer at 95C for 5 mins
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80

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

Preparation of Protein Lysates from Cells - Revision history

Iharvey at 21:43, 5 January 2018

Iharvey at 18:33, 15 December 2017

Iharvey: Created page with "==Materials== *RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. *Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells..."

Iharvey: Created page with "==Materials== RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells..."