Difference between revisions of "Preparation of Protein Lysates from Cells"
From Bridges Lab Protocols
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#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 | + | #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 | ||
Latest revision as of 21:43, 5 January 2018
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 85C for 2-3 mins
- Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
