Jacobs:Protocol Total Protein Isolation using RIPA buffer

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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.

  • Cell Scraper
  • 1.5 ml microcentrifuge tube
  • Centrifuge
  • Pipette man
  • Pipette tips

Procedure

  1. Carfully 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. Add 0.5 ml 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. (Perform Steps 1-2 in fume hood)
  5. Transfer lysate to 1.5 ml microcentrifuge tube.
  6. Allow samples to stand for 5 mins at 4C.
  7. Centrifuge the resulting mixture at 14,000g for 15 mins at 4C. This separates the total protein (supernatant) from the cellular debris (pellet)
  8. Transfer supernatant to a new tube for further analysis


Contact

  • Originally prepared by CRJ-EJC 12/30/06


or instead, discuss this protocol.

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