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| This protocol describes how to produce a nuclear extract and a cytoplasmic extract from dounce homogenized HeLa cells. |
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1. Prepare Buffer A and Buffer C, make sure rotors are available and at 4°C, and all containers are clean. This protocol requires sterile tissue culture equipment and sterile technique.
2. Grow HeLa cells to 9 X 105cells/ml in SMEM Media in 3 liters per flask (see Hint #2). Measure by counting the cells with a hemacytometer.
3. Dilute the cells with 2 to 2.5 liters of SMEM media per flask, bringing the final volume to 5 to 5.5 liters per flask.
4. Grow the cells overnight in incubator (see Protocol for Growth of Tissue Culture Cells).
5. Measure the number of cells/ml again and record the total cell numbers to calculate how much Buffer C to prepare. All subsequent steps are on ice and with solutions and equipment cooled to 4°C.
6. Harvest the cells in 500 ml centrifuge bottles for 5 min at 2,500 X g. After spinning, decant the supernatant into a dilute bleach solution for proper disposal.
7. Wash the cells: Resuspend each cell pellet in 10 ml of 4°C PBS. Collect into one bottle. Then rinse empty bottles with PBS and add to the one bottle containing the resuspended cells. The final consolidation can be split into two tubes.
8. Pellet the cells again at 2,500 X g for 6 min.
9. Note the Packed Cell Volume (PCV) by comparing to a volume of water in an empty tube.
10. Discard the supernatant, and add 5X PCV (5 times the packed cell volume equivalent) of Buffer A plus DTT and PMSF to each tube.
11. Seal the tubes and shake vigorously to resuspend cell pellet.
12. Let stand on ice for 15 min.
13. Centrifuge for 8 min at 2,000 X g.
14. Aspirate away the supernatant and resuspend the cell pellet in 2X PCV of Buffer A. Seal bottle and shake vigorously.
15. Lyse the resuspended cells using the dounce homogenizer with pestle "B" by transferring enough extract to fill the dounce homogenizer (without pestle inserted) to the top of the cylindrical area but below the spherical area. Apply 14 strokes and check extent of lysis by comparing 2 μl of pre- and post-dounce cell suspension by light microscopy. More douncing is required if more than 20% of the cells are still intact.
16. After sufficient lysis has occurred, collect lysed cells into centrifuge tubes and centrifuge the tubes at 1,000 X g for 10 min.
17. Slowly and carefully use a pipette to transfer the supernatant to 50 ml polypropylene conical tubes and store on ice. This is the cytoplasmic fraction.
18. Re-centrifuge the pellets 4,000 X g for 10 min and use a pipette to transfer remaining supernatant to the 50 ml conical tubes on ice.
19. Freeze the cytoplasmic fractions by placing the 50 ml conical tubes in a dry ice/ethanol bath for 10 min followed by storage long-term at -80°C.
20. Resuspend the pellet in 3 ml of Buffer C plus DTT and PMSF per 1 X 109 cells.
21. Shake the tube vigorously to resuspend nuclei in Buffer C.
22. Incubate the nuclei suspension at 4°C for 30 min with continual mixing on a "nutator" or other rotary device.
23. After incubation, centrifuge extracts at 17,000 X g for 15 min.
24. Use a pipette to transfer the supernatant to 50 ml propylene conical tubes and discard the pellet of cell debris.
25. Freeze the supernatant (nuclear extract) in the Dry Ice/Ethanol bath for 10 min, and store the extract at -80°C.
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| PMSF |
| Store at -20°C.
0.1M PMSF in Isopropanol.
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| DTT |
| 1M DTT in ddH2O
Store at -20°C.
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| PBS |
| 2.7 mM KCl
pH 7.2.
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
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| SMEM Media |
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| Buffer D |
| 0.5 mM PMSF
100 mM KCl
1 mM DTT
0.2 mM EDTA
10% (v/v) Glycerol
Store at 4°C
Add DTT and PMSF fresh before use.
20 mM HEPES, pH 7.9
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| Buffer C |
| Add DTT and PMSF fresh before use.
1.5 mM MgCl2
1 mM DTT
Store at 4°C
420 mM NaCl
0.5 mM PMSF
25% (v/v) Glycerol
0.2 mM EDTA
20 mM HEPES, pH 7.9
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| Buffer A |
| 1 mM DTT
0.5 mM PMSF (CAUTION! see Hint #1)
10 mM KCl
10 mM HEPES, pH 7.9
Store at 4°C
1.5 mM MgCl2
Add DTT and PMSF fresh before use.
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HEPES
PMSF
Potassium Phosphate, Monobasic
EDTA
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
DTT
Glycerol
Magnesium Chloride
Isopropanol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. In order to obtain enough nuclear extract and given the involved process of preparing HeLa cell nuclei, large amounts of cell culture are recommended. Typically, tens of liters are necessary to produce tens of milliliters of nuclear extract. The rate of consumption of nuclear extract should be determined before deciding on the scale of this experiment.
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