Nucleolar Isolation Protocol-2
7. Resuspend the pellet with 3 ml S1 solution (Figure 3). The pellet should be resuspended readily by pipetting up and down. A pellet that cannot be resuspended contains lysed nuclei and should be discarded. Layer the resuspended pellet over 3 ml of S2 solution. Take care to keep the two layers cleanly separated. Centrifuge at 1430g (2500 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) for 5 min at 4°C. This step results in a cleaner nuclear pellet (Figure 3). Resuspend the pellet with 3 ml of S2 solution by pipetting up and down.
|
Figure 3. Step 7 of the procedure. Note the clear boundary between S1 and S2 layers before centrifugation. Insets show the DIC images of the supernatant and pellet. Note the prominent nucleoli inside the nuclei in the pellet. Bars: 10µm. |
8. Sonicate the nuclear suspension for 6 x 10 second bursts (with 10 second intervals between each burst) using a Misonix XL 2020 sonicator fitted with a microtip probe and set at power setting 5 (Figure 4 Left). Check the sonicated nuclei under a phase contrast microscope. There should be virtually no intact cells and the nucleoli should be readily observed as dense, refractile bodies (Figure 4 right). The optimal sonication time depends on the cell type used. If you attempt to isolate nucleoli from a cell type from the first time, it is necessary to check the sonicated material under a microscope after every 10 seconds of sonication. Over-sonication leads to destruction of nucleoli.
|
Figure 4. Left. Setup for sonication. Right. DIC image of sonicated nuclei. Note the presence of prominent nucleoli. Bar: 10µm. |
9. Layer the sonicated sample over 3 ml of S3 solution and centrifuge at 3000g (3500 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) for 10 min at 4°C (Figure 5). The pellet contains the nucleoli, whilst the supernatant can be retained as the “nucleoplasmic fraction” (Figure 5).
|
Figure 5: Step 9 of the procedure. Note the clear boundary between S2 and S3 layers before and after centrifugation. The pellet should be small but visible. Insets show DIC images of the supernatant and pellet. The pellet should contain purified nucleoli. Bars: Left inset: 10µm, right inset: 20µm. |
10. Resuspend the nucleoli with 0.5 ml of S2 solution, followed by centrifugation at 1430 (2500 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) for 5 min at 4°C. The pellet contains highly purified nucleoli. Check under a phase contrast microscope to ensure this preparation contains only highly purified nucleoli without any other material (Fig 5). The nucleoli can be resuspended in 0.5ml of S2 solution and stored at –80oC.
Notes
(1) Making 2.55M sucrose stock
Here is a protocol for preparing a sucrose stock solution (Cline and Ryel,l 1971) suitable for the nucleolar isolation protocol. The resulting solution is 2.55M, or 66% by weight. Its density is 1.3224g/cm3 at 20oC, and refractive index is1.4558. The stock solution is stable indefinitely at 4oC. This procedure can be carried out at RT. There is no need to heat up the solution to help dissolving the sucrose. Heating up an incompletely dissolved sucrose solution can lead to charring of sucrose and affect the quality of the sucrose solution.
1. Weigh out 1710 g sucrose (BDH). Keep it aside in a clean container.
2. Put exactly 900ml water and a magnetic bar in a 5 litre beaker. Put the beaker on a stirrer and start stirring.
3. Add 1/3 of the sucrose into the beaker. Make sure the magnetic bar is rotating freely. Stir for 1 hour.
4. Add another 1/3 of the sucrose into the solution. Again make sure the rotation of the stir bar is not impaired. Stir for another 1 hour.
5. Add the remaining sucrose. Stir for another 1 hour, or until all the sucrose has gone into solution. The final volume should be exactly 2 litres.
(2) Sonication
We use a Misonix 2020 sonicator fitted with a microtip at power setting 5. To ensure reproducible soncation these points should be followed:
- It is necessary to tune the sonicator every time after you change the probe. Follow the manufacturer’s manual for the tuning procedure.
- Sonication produces intense and localized heat in your solution. If you are concerned about the heating, the correct way to reduce heating is to shorten the sonication time and to increase the intermission between bursts. Keeping the tube on ice or performing the sonication in the cold room is helpful, but is not the most effective way of heat control.
- If the probe is too close to the liquid surface, it produces a foam and reduces the efficiency of sonication. Make sure the probe is well submerged in the solution, about 5mm above the bottom of the tube. Do not, however, touch the bottom or the wall of the tube with the probe.
- Sonicator probe that has been used repeatedly develops pits on its end. The sonication efficiency gradually decreases as time goes on. Therefore, the sonication time reecommended here can only be used as guideline. Always monitor the outcome of sonication using a phase contrast microscope. You may need to adjust the sonication time to maintain the efficiency especially if the probe is getting old. Change the probe when the efficiency is noticeably down.
(3) Analysis of the isolated nucleoli
- To immunolabel the purified nucleoli, spot about 5 µl of the nucleolar suspension on to a polylysine-coated slide (BDH Cat no: 406/0178/00), and air dry the spot. Rehydrate the slide in PBS for 5 min before carrying out a standard immunostaining procedure.
- To separate nucleolar proteins on a gel, either resuspend directly in Laemmli SDS sample buffer or in your preferred buffer. The high concentration of nucleic acid in the isolated nucleoli makes the lysed sample very viscous. The sample can be clarified by passing through a QIAshredder spin column (Qiagen Cat no: 79654). Nucleoli can also be extracted with RIPA buffer (150 mM NaCl,1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, COMPLETE protease inhibitor cocktail). Immunoprecipitations can be performed from nucleolar lysates prepared in RIPA buffer.
(4) Adapting nucleolar isolation protocol to use with other cell types
The above protocol can readily be adapted to other cell types. Apart from HeLa cells, we have used this protocol, with minor modifications, to isolated nucleoli from MCF-7 (human breast epithelium), WI-38 (human fibroblast), IMR-32 (human neuroblastoma), HL60 (human promyelocytic leukemia) and plant Arabidopsis thalina cells. When adapting the protocol to a different cell type, make sure you control each step by carefully checking the products after each step under a phase contrast microscope. For example, different cell types may require a different homogenization (step 4) and/or sonication strength (step 7). The concentration of MgCl2 also appears crucial to the purity of the isolated nucleoli. If the isolated nucleoli are not pure enough, try lowering the concentration of MgCl2 in the S2 and S3 solutions. If the yield is poor, or if the nucleoli look fragmented, use more MgCl2.
