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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..."
==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]]
*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]]