Nuclear Extract Protocol

Before Beginning

  • Make sure that you have access to a microcentrifuge that is at 4ºC.
  • Obtain special Ultracentrifuge 1.5 mL tubes
  • Keep on Ice
  • Reminder ­ Wear Gloves, lab coat and eye protection!!!

Instructions

  1. Treat Cells accordingly.  If LIF, 50-100 ng/mL for 20 minutes.
  2. Aspirate media from plates.  Wash 1 time in ice cold 1 x PBS.  Keep on ice during all steps.
  3. Add 400 uL NuEx Lysis Buffer supplemented with Protease and phosphatase inhibitors (PMSF, aprotinin, leupeptin, pepstatin, DTT, NaF, NaVO) to the surface of the plate.
    1. If using tissue, add 1mL of Buffer per 100mg of tissue.
  4. Scrape cells quickly while on ice.
  5. Collect lysate into special ultracentrifuge tubes.
  6. Spin at 4ºC at 4000 rpm in a tabletop microfuge for 5 minutes.
  7. Remove supernatant ­ this is your cytosolic fraction.
  8. Resuspend Pellet gently, by hand (do not use pipet tip or vortex) in 400 uL of low salt buffer supplemented with inhibitors.
  9. Spin at 4ºC, 4000 rpm for 5 minutes.
  10. Resuspend nuclear pellet in a buffer (high salt + inhibitor cocktail) volume that is equal to the size of the pellt (~10 to 20 uL) by vortexing hard.  Let sit on ice for 45 minutes, vortexing occaisionally.
  11. Spin in tabletop ultra at 40,000 rpm for an hour at 4ºC (rotor is kept in 4ºC).
  12. Store at -80ºC.

Lysis Buffer (for 50 mL)

  • 0.5mL of 1M Hepes, pH 7.9 (10mM)
  • 0.166mL of 3 M KCl (10mM)
  • 1mL of 0.1 M MgCl2 (2mM)
  • 0.25mL of 100% NP-40
  • 46.8mL of ddH2O

Low Salt Solution (for 10mL)

  • 200uL of 1 M Hepes, pH 7.9 (20mM)
  • 2.5mL Glycerol (25%)
  • 20uL of 1 M MgCl2 (2mM)
  • 0.33mL of 3 M KCl (0.1mM)
  • 20uL of 0.5 M EDTA (1mM)
  • 6.49mL of ddH2O