Jacobs:ProtocolTotalProteinIsolationUsingRIPALysisBuffer

Materials

• RIPA buffer (RIPA buffer enables the extraction of cytoplasmic, membrane and nuclear proteins and is compatible with many applications, including reporter assays, protein assays, immunoassays and protein purification. RIPA Buffer does not contain protease or phosphatase inhibitors. However, if desired, protease and phosphatase inhibitors can be added to the RIPA buffer just before use to prevent proteolysis and maintain phosphorylation of proteins.)

• PMSF (phenylmethanesulphonylfluoride, a serine protease inhibitor)

• Sodium orthovanadate (inhibitor of protein tyrosine phosphatases, alkaline phosphatases and a number of ATPases, most likely acting as a phosphate analogue, the VO43- ion binds irreversibly to the active sites of most protein tyrosine phosphatases)

• Protease inhibitor cocktail (proprietary mixture)

• 1X PBS

• Cell scraper

• 1.5 ml microcentrifuge tube

• Centrifuge

• Pipette man

• Filtered pipette tips

• Kimwipes

• Waste beaker

Procedure

1. Each group will receive one tissue culture dish containing 3T3 fibroblasts

2. Label 2 microcentrifuge tubes: Protein Group#

3. One person will prepare RIPA Lysis buffer for the entire class:

1. Add 10 μL PMSF solution, 10 μL sodium orthovanadate solution and 10 μL protease inhibitor cocktail solution to 1ml of 1X RIPA buffer to prepare complete RIPA Lysis buffer

4. Pour off media from tissue culture dish into waste container

5. Wash cells twice with PBS pouring excess off into waste beaker

6. Carefully soak up any extra PBS with a Kimwipe

7. Add 150ul of RIPA lysis buffer to the culture dish

8. Use cell scraper to scrape cells from the bottom of the dish

9. Pass cell lysate through pipette 20 times to form homogeneous lysate

10. Transfer lysate to 1.5 ml microcentrifuge tube

11. Allow samples to stand for 5 mins at 4C (cold room)

12. Centrifuge the resulting mixture at 14,000g for 15 mins at 4C to separate cell debris from protein

13. Transfer supernatant to a new tube and store at -20C