Difference between revisions of "Preparation of Protein Lysates from Cells"
From Bridges Lab Protocols
(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 | ||
Revision as of 18:33, 15 December 2017
Materials
- RIPA Buffer (see 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 95C for 5 mins
- Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
