View source for Preparation of Protein Lysates from Cells

==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 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.  
#Heat samples with loading buffer at 85C for 2-3 mins
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80


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