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Total Procedure

1. Prepare RIPA Lysis buffer by adding PMSF or your own desired  protease inhibitors to the solution
2. Pour off media from tissue culture dish into waste container
3. Wash cells twice with PBS pouring excess off into waste beaker
4. Carefully soak up any extra PBS with an absorbent.
5. Add 500 ul of RIPA lysis buffer to the culture dish.() 
6. Use cell scraper to scrape cells from the bottom of the dish.
7. Pass cell lysate through pipette 20 times or by vortexing to form homogeneous lysate.
8. Transfer lysate to 1.5 ml microcentrifuge tube.
9. Allow samples to stand for 5 mins in ice or at 4^oC (cold room).
10. (Optional) repeat step 7-9 againe.
11. Centrifuge the resulting mixture at 14,000g for 15 mins in ice or at 4^oC to separate cell debris from protein.
12. Transfer supernatant to a new tube and store at -20^o C.