Preparation of Protein Lysates from Cells: Difference between revisions
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..." |
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#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 | ||
#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 | |||
#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 | ||