Nuclear/Cyto
Extraction
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!!!
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.
3a. 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