Preparation of Protein Lysates from Cells

Materials

• RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors.
• Cells (fresh or frozen)

Protocol

1. Cool centrifuge to 4C
2. If cells are not already frozen in buffer, add ~400ul RIPA plus PI while keeping cells on ice
3. Scrape cells and transfer to cold micro centrifuge tube on ice
4. Incubate on ice for 15 minutes
5. Centrifuge at 14 000 RPM at 4C for 10 min
6. Remove supernatant to clean tube. If lysing fat cells, try to avoid the floating fat cake. If necessary respin to clarify
7. Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.
8. Heat samples with loading buffer at 95C for 5 mins
9. Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80