Jacobs:Protocol Total Protein Isolation using RIPA buffer 2

Materials

• PBS
• RIPA buffer
• PMSF
• Sodium orthovanadate solution
• Protease inhibitor cocktail solution
• Cell Scraper
• 1.5 ml microcentrifuge tube
• Centrifuge
• Pipetteman/Pipette tips
• Pipet/Pipet aid

Procedure

1. Carefully remove culture media from adhered cells
2. Wash cells twice with cold PBS
3. RIPA buffer does not contain any protease or phosphotase inhibitors. If desired combine 10 µl PMSF solution, 10 µl sodium orthovanadate solution and 10-20 µl protease inhibitor cocktail solution (Santa Cruz Biotechnology) per ml of 1X RIPA Lysis buffer to prepare complete RIPA. Note: This should be done immediately before applying to cells
4. Lyse cells directly on culture dish by adding 250ul of RIPA lysis buffer (for up to 5x106 cells). Use cell scraper to scrape off cells and pass cell lysate through pipette 20 times to form homogeneous lysate.
5. Transfer lysate to 1.5 ml microcentrifuge tube.
6. Allow samples to stand for 15 mins at 4ºC.
7. Centrifuge the resulting mixture at 14,000g for 15 mins at 4ºC. This separates the total protein (supernatant) from the cellular debris (pellet).
8. Transfer supernatant to a new tube for further analysis.

Bradford Assay – to determine total protein concentration

1. Standard: pipette 0, 2, 4, 6, and 10 l of BSA (2 mg/ml) into assigned wells of a 96-well plate. Add water to make 10 l total volume.
2. Pipette 10 l of unknown samples into individual wells of 96-well plate.
3. Add 200 l of Bradford Reagent into all wells containing standard or sample
4. Read absorbance at 595 nm without prior incubation (concentration [g/l])

or instead, discuss this protocol.