Protocols

Nuclear Extract

Nuclear Extraction (adapted from Mirmira Lab, University of Virginia)

Materials:

• Mammalian cells (100 mm dish, adherent culture)
• Ice
• Cold PBS
• Cold Buffer A
• Cold Buffer B
• Halt protease and phosphatase inhibitor single-use cocktail (100X) (Thermo Scientific #78442)
• 10% IGEPAL CA-630
• 1 M Dithiothreitol (DTT)

Recipes:

Buffer A (enough for 10 plates; store at 4^oC up to 1 month)

Buffer B (enough for 10 plates; store at 4^oC up to 1 month)

Procedures:

Culture adherent cells to approximately 80% confluence on 100mm polystyrene tissue culture plates. Cells should be in log phase growth and healthy.

1. Add the following to 5 ml Buffer A:
   • 50 µl Halt protease and phosphatase inhibitors
   • 200 µl 10% IGEPAL CA-630
   • 5 µl 1M DTT
2. Aspirate or decant media
3. Wash cell monolayer gently twice with 10 ml cold PBS. Aspirate or decant excess PBS
4. Add 0.5 ml of Buffer A with inhibitors, IGEPAL, and DTT to each plate and swirl to distribute buffer
5. Incubate at room temperature (RT) for 10 minutes
6. Using a cell scraper or silicone spatula, scrape the cells and pipet up and down with P1000 several times to disrupt cell clumps
7. Transfer the lysate to 1.5 ml microcentrifuge tubes
8. Centrifuge at 4ºC at top speed (15,000 X g) for 3 minutes
9. Remove supernatant. Save supernatant (cytosolic fraction), if desired; otherwise, discard
10. Add the following to 2 ml of Buffer B:
    • 20 µl Halt protease and phosphatase inhibitors
    • 2 µl DTT
11. Re-suspend each pellet by adding 150 µl of Buffer B with inhibitors and DTT. Pipette up and down with a P200
12. Place the tubes on ice for 2 hours. During the two hour incubation, vortex the tubes every 15 minutes
13. Centrifuge at 4ºC at top speed (15,000 X g) for 5 minutes
14. Remove and pool supernatants into a fresh tube
15. Determine protein concentration by the bicinchoninic acid method (Pierce 23228) or other preferred method
16. Aliquot and store extract at -80ºC or -20ºC avoiding multiple freeze/thaw cycles