Small scale Nuclear Protein Extraction without the use of a detergent

Xiaobing Gozani Lab 8/05

Small scale Nuclear Protein Extraction without the use of a detergent

Note: The Sigma Nuclear Extract procedure requires at least 100 μl of PCV (packed cell volume). Use of a syringe is recommended for small-scale preparations (0.1-1 ml). Passage of more than one millliliter through a syringe be difficult due to the needle gauge size.

1. Scrape adherent cells using fresh PBS and move into an appropriate conical centrifuge tube.

2. Centrifuge for 5 minutes at 450 x g. 2.

3. Estimate the packed cell volume (PCV, probably ~200 μl).

4. Add 5X PCV (e.g. 1mL) of 1X Lysis Buffer (including DTT and protease inhibitors) to 200 μl of PCV. Resuspend the cell pellet gently. Avoid foam/bubbles.

5. Incubate the packed cells in lysis buffer on ice for 15 min. Cells will swell.

6. Centrifuge the suspended cells for 5 minutes at 420 x g.

7. Resuspend the packed cells in 400 μl (2X PCV) of the 1X Lysis Buffer.

8. Cell disruption.

1. Using a glass tissue homogenizer, transfer the cells into a glass tissue grinding tube (e.g. Dounce). Grind on ice slowly with five up-and-down strokes using a type B pestle. Avoid foam/bubbles. OR

2. Using a syringe with a narrow-gauge (No. 27) hypodermic needle, fill the syringe with 1X Lysis Buffer. The syringe plunger is used to displace the buffer as fully as possible. This removes all the air from the syringe and prevents excess air being pumped into the cell suspension during lysis. Draw the cell suspension slowly into the syringe and then eject with a single rapid stroke. Repeat five times. Note: The number of strokes needed varies between cell lines. Start with 5 strokes and then check lysis under the microscope. Lysis should be 80-90%. Lysis can be observed by the addition of the Trypan Blue solution to an aliquot of cells. The dye is excluded from the intact cells, but stains the nuclei of lysed cells.

9. Centrifuge the disrupted cells in suspension for 20 min at 10,000–11,000 x g.

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

11. Resuspend the crude nuclei pellet in ~140 μl (2/3X PCV) of Extraction Buffer (0.42mM salt) containing DTT and protease inhibitors. If the procedure is being performed with a homogenizer, it is recommended to give 10 more strokes at this point. Note: The salt concentration in the Extraction Buffer is 0.42 M, a commonly used extraction condition. In rare cases a lower or a higher salt concentration may be needed to more effectively extract a particular protein. In that case, dilute the Extraction Buffer with the 1X Dilution and Equilibration Buffer or add NaCl to the Extraction Buffer to reach the desired salt concentration.

12. Shake gently for 30 minutes.

13. Centrifuge for 5 minutes at 20,000-21,000 x g.

14. Transfer the supernatant to a clean, chilled tube.

15. Snap-freeze the supernatant in aliquots with liquid nitrogen and store at –70 °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.