Nuclear Extracts-Adherent Cells
Michael S. Parmacek
Ref: Digham et al., NAR 11:1475-1489, 1983
Reagents:
1. 100 mM PMSF in 100% EtOH Make Fresh-(0.44 g/25 ml EtOH)
2. Buffer A (50ml): 10 mM Hepes pH 7.9 500 ul 1M Hepes pH 7.9
1.5 mM MgCl2 75 ul 1M MgCl2
10 mM KCl 500 ul 1M KCl
0.5 mM DTT 25 ul 1M DTT
0.5 mM PMSF 250 ul of 100 mM PMSF (add just prior to use)
48.9 ml ddH20
3. Buffer C (50ml) 20 mM Hepes pH 7.9 1 ml 1M Hepes pH 7.9
25% glycerol 12.5 ml glycerol
.42 M KCl 21 ml 1M KCl
1.5 mM MgCl2 75 ul 1M MgCl2
0.2 mM EDTA 20 ul 500 mM EDTA
0.5 mM DTT 25 ul 1M DTT
0.5 mM PMSF 250 ul 100 mM PMSF (add just prior to use)
15 ml ddH20
4. Buffer D (1L) 20 mM Hepes pH 7.9 20 ml 1M Hepes pH 7.9
20% glycerol 200 ml glycerol
0.1 M KCl 100 ml 1M KCl
0.2 mM EDTA 400 ul 500 mM EDTA
0.5 mM DTT 0.5 ml 1M DTT
0.5 mM PMSF 5 ml 100 mM PMSF (add just prior to use)
674 ml ddH20
ALL BUFFERS AND ROTORS AND TUBES MUST BE PRE-COOLED TO 4°C!!
METHOD:
1. Trypsinize (9) T150 flasks of cells per standard protocol.
2. Resuspend cells in DMEM, 10% FCS (can use C2 grow) to inactivate trypsin and spin 1500 RPM, 4°C, 7 min.
3. Wash 2 times with PBS (containing Ca and Mg) and spin down as above in step 2.
4. Resuspend the pellet in 4 packed cell volumes of Buffer A and place on ice 10 minutes.
5. Spin 1500 RPM, 4°C, 7 min.
6. Resuspend the pellet in 2 packed cell volumes of Buffer A and transfer to a pre-cooled Dounce homogenizer.
7. For C2C12 myotubes (this varies for different cells), stroke 10 times with a B pestle and 4 times with an A pestle.
8. To 2 ul of cells and 20 ul of PBS add 5 ul trypan blue and check for intact nuclei on the haemocytometer ((85-90% of cells should be lysed)
9. Transfer to pre-cooled Oak Ridge tube and spin in JA13.1 rotor, 4°C, 20 min.
10. Discard the supernatant and resuspend the pellet in 1-1.5 packed cell volume of Buffer C.
11. For C2C12 myotubes (this varies for different cells), Dounce 10 times with pestle B and 10 times with pestle A. (for 3T3 and C2 blasts can dounce 5 times with pestle A)
12. Transfer to pre-cooled Oak Ridge tube and place on rocking platform at 4°C for 30 minutes.
13. Inspect nuclei with trypan blue, (should have destroyed most nuclei with pale blue background)
14. Spin at 12,500 RPM, 4°C, 30 minutes in a pre-cooled JA17 rotor (DO NOT SPIN IN A SARSTEDT TUBE)
15. Dialyze the supernatant in 1L of Buffer D, 4°C for 2hrs.
16 Repeat dialysis for 2 hours.
17. Place nuclear extract in Oak Ridge tube and centrifuge in a JA17 rotor, 12500 RPM, 30 min, 4°C.
18. Aliquot the nuclear extracts in pre-cooled micrfuge tubes and quick freeze in a dry ice ethanol bath. Store at -70°C.
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