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
Each group will receive one tissue culture dish containing 3T3 fibroblasts
Label 2 microcentrifuge tubes: Protein Group#
One person will prepare RIPA Lysis buffer for the entire class:
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
Pour off media from tissue culture dish into waste container
Wash cells twice with PBS pouring excess off into waste beaker
Carefully soak up any extra PBS with a Kimwipe
Add 150ul of RIPA lysis buffer to the culture dish
Use cell scraper to scrape cells from the bottom of the dish
Pass cell lysate through pipette 20 times to form homogeneous lysate
Transfer lysate to 1.5 ml microcentrifuge tube
Allow samples to stand for 5 mins at 4C (cold room)
Centrifuge the resulting mixture at 14,000g for 15 mins at 4C to separate cell debris from protein
Transfer supernatant to a new tube and store at -20C
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