Nuclear Protein Extraction Without the Use of Detergent

Detergents can interfere with the labeling efficiency of the extracted proteins. Therefore, a procedure that does not include the use of detergents should be used for the preparation of nuclear proteins.

The CellLytic™ NuCLEAR™ Extraction Kit (Product No. NXTRACT) may be used for preparation of nuclear protein. Be certain to use the procedure that omits the use of a detergent. A nuclear protein extract prepared with this kit requires dialysis to adjust the pH prior to labeling with the Cy dyes.

The following procedure is for protein extraction from 200 µL of packed cell volume (PCV) and represents the non-detergent procedure in the kit. For different packed cell volumes, calculate accordingly.

Note: The following procedure describes the preparation of crude nuclear extracts using a syringe or a glass tissue homogenizer. The procedure requires at least 100 µL of PCV. Use of a syringe is recommended for small-scale preparations (0.1-1 mL). Passage of more than 1 mL through a syringe may cause difficulties due to the needle gauge size.

Extraction from Cells

1. Prepare a fresh solution of 0.1M DTT with ultrapure sterile water.
2. Prepare Lysis Buffer:
   • hypotonic: 10 mM HEPES, pH 7.9, with 1.5 mM MgCl₂ and 10 mM KCl.
   • isotonic (or protein extraction from fragile cells): 10 mM Tris HCl, pH 7.5, with 2 mM MgCl₂, 3 mM CaCl₂, 0.3 M Sucrose.
3. To 1,400 µL of Lysis Buffer (hypotonic or isotonic), add 14 µL of the 0.1 M DTT solution and 14 µL of the Protease Inhibitor Cocktail.
4. Prepare Extraction Buffer: 20 mM HEPES, pH 7.9, with 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) Glycerol.

From Adherent Cells

1. Grow cells to 70-80% confluency.
2. Remove the growth medium from the cells.
3. Rinse the cells twice with PBS, being careful not to dislodge any cells.
4. Discard the PBS. Scrape the cells using fresh PBS and collect into an appropriate conical centrifuge tube.
5. Centrifuge for 5 minutes at 450 x g.
6. Decant and discard the supernatant.
7. Estimate the packed cell volume (PCV).
8. Proceed to step 7 of the procedure for Non-adherent cells.

From Non-Adherent Cells

1. Collect the cells into an appropriate centrifuge conical tube.
2. Centrifuge for 5 minutes at 450 x g.
3. Decant and discard the supernatant.
4. Wash cells twice by resuspending the cell pellets in PBS and centrifuge for 5 minutes at 450 x g.
5. Decant and discard supernatant.
6. Estimate the packed cell volume (PCV).
7. Add 1 mL (5X PCV) of Lysis Buffer (including DTT and Protease Inhibitors) to 200 µL of PCV.
8. Resuspend the cell pellet gently. Avoid foam formation. If working with small volumes, the suspended cells may be transferred to a microcentrifuge tube.
9. Incubate the packed cells in Lysis buffer for 15 minutes, allowing cells to swell.
10. Centrifuge the suspended cells for 5 minutes at 420 x g. Decant supernatant and resuspend the pellet of packed cells in 400 µL (2X PCV) of the Lysis Buffer.
11. Using a glass tissue homogenizer, transfer the cells into a glass tissue grind tube. Grind on ice slowly with five up-and-down strokes using a type B pestle. Avoid foam formation.
    OR
    Using a syringe with a narrow-gauge (No. 27) hypodermic needle, fill the syringe with 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 (using the tissue homogenizer or the syringe) varies between cell lines. Start with 5 strokes
    and then check lysis under the microscope. Lysis should be 80-90%. If the lysis is not sufficient, perform several more
    strokes until lysis is complete.
    • Lysis can be observed by the addition of a Trypan Blue solution to an aliquot of cells. The dye is excluded from the intact
    cells, but stains the nuclei of lysed cells. If nuclear lysis or clumps of nuclei are visualized, or if a gelatinous mass is
    observed, the cell disruption may have been too vigorous or too many strokes were performed.
12. Centrifuge the disrupted cells in suspension for 20 minutes at 10,000–11,000 x g.
13. Transfer the supernatant to a fresh tube. This fraction is the cytoplasmic fraction.
14. Add 1.5 µL of the 0.1 M DTT solution and 1.5 µL of the Protease Inhibitor Cocktail to 147 µl of the Extraction Buffer.
15. Resuspend the crude nuclei pellet in ~140 µL (2/3X PCV) of Extraction Buffer containing DTT and Protease Inhibitors. If the procedure is being performed with a tissue homogenizer, it is recommended to give 10 more strokes at this point.
16. Shake gently for 30 minutes.
17. Centrifuge for 5 minutes at 20,000-21,000 x g.
18. Transfer the supernatant to a clean, chilled tube.
19. Continue with dialysis or store at –70 °C.

Extraction from tissue

The following procedure is for extraction of nuclear proteins from 100 mg of tissue. For different tissue weight, calculate accordingly.

1. Prepare a fresh solution of 0.1M DTT with ultrapure sterile water.
2. Prepare Lysis Buffer
   a. Prepare a fresh solution of 0.1M DTT with ultrapure sterile water.
   b. Prepare Lysis Buffer:
   • hypotonic: 10 mM HEPES, pH 7.9, with 1.5 mM MgCl₂ and 10 mM KCl.
   • isotonic (or protein extraction from fragile cells): 10 mM Tris HCl, pH 7.5, with 2 mM MgCl₂, 3 mM CaCl₂, 0.3 M Sucrose.
   c. To 1,400 µL of Lysis Buffer (hypotonic or isotonic), add 14 µL of the 0.1 M DTT solution and 14 µL of the Protease
   Inhibitor Cocktail.
   Note: For tissues we tested, the hypotonic buffer worked better than the isotonic. If the tissue is found to be too fragile, one can use the isotonic Lysis Buffer.
3. Prepare Extraction Buffer
   a. Prepare Extraction Buffer: 20 mM HEPES, pH 7.9, with 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) Glycerol.
   b. Add 1.5 µL of the prepared 0.1 M DTT solution and 1.5 µL of the protease inhibitor cocktail to 147 µL of the Extraction Buffer.
4. Rinse the tissue twice with PBS buffer. Discard the PBS.
5. Resuspend the tissue gently in 1 mL (5X PCV) of the Lysis Buffer containing DTT and protease inhibitors.
6. Homogenize the tissue (using the tissue homogenizer) until more than 90% of the cells are broken and nuclei are visualized under the microscope.
7. Centrifuge the disrupted cells for 20 minutes at 10,000–11,000 x g.
8. Transfer the supernatant to a fresh tube. This fraction is the cytoplasmic fraction.
9. Resuspend the crude nuclei pellet in ~140 µL (2/3X PCV) of Extraction Buffer containing DTT and Protease Inhibitor. At this stage a short homogenization can be performed to facilitate nuclear extraction.
10. Shake gently for 30 minutes.
11. Centrifuge for 5 minutes at 20,000 – 21,000 x g.
12. Transfer the supernatant to a clean, chilled tube.
13. Continue with dialysis or store at –70 °C.

Dialysis

Before performing the labeling procedure it is important to bring the extract to the required pH by dialysis.

1. Prepare 0.1M, pH 9.5-9.6, Carbonate-Bicarbonate Buffer (dissolve 2 capsules of Product No. C3041 into 100 mL water).
2. Dialyze at 4 °C for 2 hours in a dialysis buffer volume 1000 times the volume of the nuclear protein extract.
3. Replace the dialysis buffer to a freshly prepared carbonate buffer and dialyze for an additional 2 hours, at 4 °C. Determined protein concentration according to the Bradford procedure.