Large scale Nuclear Protein Extraction without the use of a detergent

Large scale (50 L) HeLa Cell Nuclear Protein Extraction

Adapted from Yuan Lab's protocol

Note: All steps, buffers, glassware are in cold room or on ice.

1. Estimate the packed cell volume (PCV), it should be ~50 ml.

2. Add 5X PCV (250 ml) of 1X hypotonic Lysis Buffer (final vol. in tube is 300 ml).This is a rapid buffer exchange step. Try to be quick, but be sue to resuspend the cells gently.

3. Quickly aliquot the cells into 50 ml conical tubes and spin at 3000 rpm for 5 min.

4. Pour off the supe. Bring the cells to 3X the original PCV with hypotonic buffer (now the final vol. should be 150-200 mls).

5. Let the cells swell on ice for 10 min.

6. Cell disruption: Using a glass tissue homogenizer, dounce on ice slowly but steadily 12 times. Check lysis by Trypan Blue staining under the microscope. Lysis should be 80-90%. If the lysis is not sufficient, perform several more strokes until lysis is complete, but avoid douncing too much because you don't want to break the nuclei.

7. Centrifuge at 4K for 15 min.

8. Transfer the supernatant to a fresh tube. This fraction is the cytoplasmic fraction.

9. The pellet contains the nuclei. Estimate the packed nuclear volume (PNV, should be bigger than PCV).

10. Add 1/2 PNV of Low Salt Buffer containing DTT and protease inhibitors to resuspend the pellets. Combine and add to a 500 ml beaker.

11. Add 1/2 PNV of High Salt Buffer (1.4 M salt) in a slow, dropwise fashion while stirring gently. Note: Add High Salt Buffer drop by drop over a 10-15 min period; the stirring should be fast enough to mix the solution, but not fast enough to damage the nuclei. The final salt conc. is 0.35 M.

12. Stir solution gently for 30 minutes.

13. Centrifuge for 30 minutes at 12K g (10 K rpm in SS34 rotor).

14. The supernatant is the nuclear extract. Transfer the supernatant to a clean, chilled tube. Aliquot into 0.5 or 1ml aliquots. Snap-freeze with liquid nitrogen and store at -80 °C.

Solutions for nuclear extraction preparation.

Note: add PMSF (final [ ] = 0.2 mM ) and DTT (final [ ] =0.5 mM DTT along with Protease Inhibitors (tablet) to all of the buffers right before use.

Hypotonic lysis Buffer

10 mM HEPES 7.9, 1.5 mM MgCl2 10 mM KCl

Low Salt Buffer

20 mM HEPES 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA 25% glycerol

High Salt buffer

20 mM HEPES 7.9, 1.5 mM MgCl2, 1.4 M KCl, 0.2 mM EDTA, 25% glycerol

Dialysis Buffer

20 mM HEPES 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol

Isotonic Cell Lysis Buffer

10 mM Tris 7.5,  2 mM MgCl2, 3 mM CaCl2 0.3 M sucrose

3 M KCl 224 g in 1L. 1 M MgCl2 101.7g in 500 ml.