| |
| This protocol describes a convenient and rapid method for preparing soluble nuclear extracts from as few as 3 X 107 mammalian cells. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. These extract are comparable to nuclear extracts prepared from large numbers of cells by conventional procedures: the extracts are able to support efficient transcription of a variety of class II promoters and they support efficient pre-mRNA splicing and allow formation of previously characterized splicing complexes. Furthermore, DNase I footprinting and gel retardation assays can be performed directly in the extracts. |
| |
1. Harvest 2.5 X 107 HeLa cells that are 80% confluent by trypsinization or by scraping with a rubber policeman (see Hint #2).
2. Centrifuge in a table-top centrifuge at 1,000 X g for 5 min at 4°C.
3. Wash cells in 30 volumes of ice-cold PBS.
4. Centrifuge cells in a table-top centrifuge at 1,000 X g for 5 min at 4°C.
5. Determine and record the packed cell volume (PCV).
6. Add 1X PCV of Buffer A to the cells. Transfer the cells to a 1.5 ml microcentrifuge tube.
7. Incubate the cells on ice for 15 min to allow the cells to swell.
8. Lyse the cells by drawing cells through a 25-guage needle: first fill a 1 ml syringe with Buffer A and expel to remove all the air from the syringe. Then, draw the cell suspension into the syringe SLOWLY and expel with MODERATE force to lyse the cells.
9. Repeat the cell lysis 3 to 5 times.
10. Assess the amount of cell lysis under the light microscope. Continue lysing the cells until at least 80 to 90% of the cells are lysed but the nuclei are still intact.
11. Pellet the nuclei by centrifuging at 12,000 X g for 20 sec at 4°C.
12. Save the supernatant in an ice-cold 1.5 ml microcentrifuge tube. This is the postnuclear extract. The pellet is the crude nuclear pellet.
13. Add two-thirds volume of the ORIGINAL PCV of Buffer C to the crude nuclear pellet.
14. Incubate the resuspended nuclear material on ice for 30 min with occasional agitation (see Hint #3).
15. Centrifuge in a microcentrifuge at full speed for 5 min at 4°C.
16. Punch a hole in the top of a 1.5 ml microcentrifuge tube. Cool the tube on ice.
17. The supernatant from Step #15 is the nuclear extract. Transfer this supernatant to the ice-cold 1.5 ml microcentrifuge tube with the hole in the tube.
18. Cut a piece of dialysis tubing with the appropriate molecular weight cutoff.
19. Place the dialysis tubing over the opening of the microcentrifuge tube and close the tube over the dialysis tubing.
20. Secure the tube upside-down to the side of a beaker.
21. Dialyze the nuclear extract against 100 volumes of Buffer D for 2 hours at 4°C.
22. Disburse the postnuclear extracts (from Step #12) into 20 μl aliquots and quick freeze in Liquid Nitrogen.
23. Disburse 10 to 15 μl aliquots of the nuclear extract (from Step #21) and quick freeze in Liquid Nitrogen. Ten plates of 80% confluent HeLa cells will yield approximately 400 μl of nuclear extract at 8 mg/ml.
24. Store aliquots at -80°C.
|
| Buffer D |
| 0.5 mM PMSF
0.1 M KCl
Add PMSF and DTT right before use
0.2 mM EDTA, pH 8.0
0.5 mM DTT
20% (v/v) Glycerol
20 mM HEPES, pH 7.9
|
 |
| Buffer C |
| 25% (v/v) Glycerol
Add PMSF and DTT right before use
0.2 mM EDTA, pH 8.0
0.42 M NaCl
0.5 mM Phenylmethylsulfonyl Floride (PMSF) (CAUTION! see Hint #1)
0.5 mM DTT
1.5 mM MgCl2
20 mM HEPES, pH 7.9
|
|
| Buffer A |
| 10 mM KCl
10 mM HEPES, pH 7.9
0.5 mM DTT
1.5 mM MgCl2
|
|
| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
|
|
|
| |
Liquid nitrogen
HEPES
PMSF
EDTA
Potassium Chloride
Sodium Chloride
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
DTT
Magnesium Chloride
Glycerol
|
| |
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. All buffers should be ice-cold and the cells should be kept at 0 to 4°C at all times during this protocol.
3. The contributor of this protocol flicks the tube every few minutes to keep the material mixed.
|
| |
1. Lee KA, Bindereif A, Green MR. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene. Anal. Tech. 1988. 5:22-31.
|
