This section describes a method for preparing nuclear extract from suspension cultures of HeLa, Namalwa, or HeLa-derived cells stably expressing f:TR (see Subheading 3.1.2.). The protocol is essentially based upon the method of Dignam (19) with a few modifications. Both HeLa S3 and HeLa-derived sublines are grown in spinner flasks containing Jokliks medium, 10% FBS, 50 IU/mL penicillin and 50 pg/mL streptomycin and maintained at a density of 2-5 x 105 cells/mL. Namalwa cells are grown in spinner flasks containing RPMI 1640 medium, 10%FBS, 50 IU/mL penicillin and 50 pg/mL streptomycin and maintained at a density of 1-2 x 106 cells/mL.
1. Six L of either HeLa or Namalwa cells in suspension cultutre (e.g., two 3000-mL spinner flasks) are grown to density of either 5 x 105 or 2 x 106 cells/mL, respectively. Centrifuge the cells in sterile 1-L plastic bottles (approx 800 mL of culture per bottle) at 3000 rpm (1850g) for 15 min in a refrigerated RT7 centrifuge (Sorvall, Newton, CT) at 4°C with the brake turned off. Carefully pour off most of the supernatant and add fresh suspension culture on top of the existing cell pellet and repeat. When all of the culture has been centrifuged, pour off the supernatant until about 50-100 mL of media remains above the pellet. Gently resuspend the cells in the remaining media and transfer into conical 250-mL centrifuge bottles (Corning Costar Corp., Cambridge, MA). Centrifuge cells at 1850g (3000 rpm) for 15 min at 4°C with the brake on.
2. All subsequent steps are performed on ice or in a 4°C cold room using prechilled buffers and pipets. Aspirate media and resuspend cell pellet in ice-cold PBS until final cell suspension volume reaches 50 mL. Transfer cell suspension to a prechilled calibrated 50-mL centrifuge tube and recentrifuge as above.
3. Using the graduations on the tube, measure the packed cell volume (pcv) (see Note 9). With the tube on ice, resuspend the cell pellet in a volume of ice-cold hypotonic buffer approx 4 times the pcv. Centrifuge at 1250g for 10 min at 4°C. Carefully remove supernatant and resuspend cell pellet in a volume of hypotonic buffer approx 2 times the pcv. Allow the cells to swell on ice for 10 min.
4. Transfer cell suspension to a Dounce homogenizer on ice. Homogenize with 10-12 up-and-down strokes using a type B pestle. Verify lysis of outer membranes (see Note 10).
5. Transfer the homogenate to a calibrated 50 mL centrifuge tube on ice. Pellet the nuclei at 3000g (4000 rpm) for 15 min at 4°C. Carefully remove supernatant and measure the nuclear pellet volume (npv) using the graduations on the tube. Resuspend the nuclear pellet in a volume of ice-cold low-salt buffer equal to 1/2 the volume of the npv.
6. Transfer the nuclei suspension to the Dounce homogenizer on ice and homogenize with six up-and-down strokes using a type B pestle.
7. In a 4°C cold room, transfer the homogenate to a prechilled 10-mL glass beaker containing 1/4-inch magnetic stir bar. While mixing the homogenate gently on a magnetic stirrer, add (in a drop-wise fashion) a volume of high-salt buffer equal to 1/2 the volume of the npv. Continue to stir for 30 min in the cold room.
8. Transfer the extracted nuclei suspension to a prechilled polycarbonate centrifuge tube (Nalgene, cat. no. 3431-2526). Using a prechilled 50.2 Ti rotor (Beckman Coulter, Fullerton, CA), centrifuge the extract at 41,000g (18,000 rpm) in a XL-90 ultracentrifuge (Beckman Coulter) at 4°C for 30 min.
9. Transfer the supernatant into dialysis tubing (prerinsed in cold BC100) and dia-lyze against 1 L cold BC100 buffer for 3-6 h at 4°C changing the buffer several times. Dialyze until the conductivity of the nuclear extract equals that of the BC100 buffer (approx 60 |iM Ho). The protein concentration of the nuclear extract prepared in this manner is typically 10 mg/mL.
10. Transfer the dialyzed nuclear extract to a prechilled centrifuge tube and flash-freeze in liquid nitrogen. Store the extract at -80°C.
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