help with centrosome isolation - any advice much appreciated! thanks (Mar/03/2005 )
I am trying to isolate centrosomes from Hela cells. But I have some problems. could you give me some help? Thanks a lot.
I followed the protocol modified from that used by Hsu in his paper: BRCA1 is associated with the centrosome during mitosis.
I met problems in step 4. I cannot sediment all the centrosomes into the sucrose cushion. after centrifuge, there were four layers. I collected them separately and western blotted gamma tubulin. I didn't see any significant difference among the upper 3 layers, but the total protein concentration was very low in the bottom layer. Does this suggest that I should increase the centrifuge speed to get most centrosomes down into the cushion? But I'm not sure how much should I increase. And I used the eppendorf fixed angle centrifugation, does this matter?
( ref: BRCA1 is associated with the centrosome during mitosis. Hsu LC, White RL.)
1. Grow cells to exponential phase.
2. Add cytochalasin D to 1ug/ml and nocodazole to 0.2uM
Incubate at 37.Cfor 1 h.
3. harvest and lysis cells
1 mM Hepes (pH 7.2),
0.5% Nonidet P-40,
0.5 mM MgCl2,
phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate).
4. centrifuge at 2,500g for 10 min (remove swollen nuclei and chromatin aggregates)
5. filter the supernatant through a 50-µm nylon mesh
6. Adjust Hepes to 10 mM,
Add DNase I to 2 units/ml
incubate for 30 min on ice.
7. Underlay the lysate with 60% sucrose solution (60% wt/wt sucrose in 10 mM Pipes, pH 7.2/0.1% Triton X-100/0.1% 2-mercaptoethanol).
Centrifuge at 10,000 ?g for 30 min to sediment centrosomes into the sucrose cushion.
8. Purify this crude centrosome preparation further by discontinuous sucrose gradient centrifugation at 120,000 ?g for 1 h.
Usually 1-3 ?107 cells were lysed in 5 ml of lysis buffer, and the cushion consisted of 0.5 ml of 60% sucrose. After centrifugation, 1.5 ml from the bottom layer was resuspended and loaded onto a discontinuous gradient consisting of 500 µl of 70%, 300 µl of 50%, and 300 µl of 40% sucrose solutions. Fractions were collected from the bottom, 200 µl per fraction, from fractions 1-7; the remaining solution ( 1 ml) was collected as fraction 8. Each fraction was diluted in 1 ml of 10 mM Pipes buffer, pH 7.2
9. Recover centrosomes by centrifugation at 15,000 rpm for 10 min in a microfuge and denatured in SDS sample buffer (62.5 mM Tris, pH 6.8/10% glycerol/2% SDS/1.4% 2-mercaptoethanol/0.001% bromophenol blue).
there are this protocol in detail....
maybe you need to use a corex tubes for your centrifugation.....
The protocol is identical to Tim's published in Vol 134 of Methods in Enzymology. The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen in liq N2.
What you need:
Sorvall with cold HB-4
Ultra with cold SW27
Some sort of gradient fractionator with a fraction collector
16 30 ml corex tubes (cold)
A lot of these solutions are w/w; Tim says that to make these weigh out sucrose and then add buffer till weight is 100g.
Wash & Lysis:
PE: 10 mM PIPES, 1mM EDTA, 8 mM BME
Make a 50X stock and pH to 7.2 with KOH
LB: 1mM Tris-HCl, 8 mM BME
Make Tris as 2M stock and pH to 8.0 with HCl
LB + 0.5% NP-40 :
(warm for 30' to 37 deg.C to ensure NP-40 has dissolved)
PBS: 130 mM NaCl, 2 mM KCl. 8 mM Na2HPO4, 2 mM KH2PO4
Make a 10X stock
Make 600 ml of
0.1X PBS, 8% (w/w) ultrapure sucrose
8% (w/w) ultrapure sucrose
LB (w/o BME): add 280 ul BME before use
LB + 0.5% NP-40 (w/o BME): add 280 ul BME before use
and put all these buffers in coldroom
Sucrose Gradients :
Use ultrapure sucrose
20% (w/w) and 62.5% (w/w) sucrose in 1x PE + 0.1% TX-100. Make 100 grams of each as follows:
Weigh out sucrose and then on the balance add 1X PE + 0.1% TX-100 till weight is 100 grams.
Just before pouring gradients add 28 ul BME/50 g
Pour gradients night before or during drug treatment (see later). I find it easiest to pour night before and store in cold.
To pour gradients - first put a 5 ml heavy sucrose pad and then pour gradient on top of that. For the prep outlined below I pour 2 gradients in SW27 tubes:
4 ml heavy sucrose pad (use 62.5% or higher).
16 ml gradient.
The centrosomes are very close to the bottom of this gradient. The pad eliminates them from entering the curve of the tube and also gives a little leeway in setting up the fractionation.
20% (w/w) Ficoll (MW 400,000) in 1X PE + 0.1% NP-40
Make up PE + 0.1% NP-40 (no BME).
Weigh out 10g Ficoll.
Add PE + 0.1 % NP-40 till total weight = 50 grams.
Stir at RT for several hours to dissolve and store cold.
Before use, add 28 ul BME
Warm up 300 ml of CHO medium to 37 deg.C and add cytochalasin B (150 ul of 10 mg/ml) and nocodazole (300 ul of 10 mg/ml)
Make sure all buffers are in coldroom, there is rocker in the coldroom and there is a good aspirator in the coldroom. Hook up a sawed off pipet to the aspirator and make sure there is a LARGE trap (at least 4L)
Add medium with drugs to 10 plates of cells. Add medium with drugs to the other 10 plates 45' later.
After 90' in drug medium process first 10 plates: bring to coldroom and wash with the following buffers:
0.1X PBS, 8% (w/w) sucrose
8% (w/w) sucrose
and then pipet on
LB + 0.5% NP-40 (10 mls/plate)
(These washes must be done rapidly - all washes should be under 1' per plate. The way I do them is to pour on the wash buffer from a beaker or grad cylinder (~ 30 mls), immediately rock back and forth and aspirate ASAP before pouring on the next wash. It is critical to do this quickly to get good lysis or you will lose most of the centrosomes in the nuclear pellet). After adding the LB + 0.5% NP-40 transfer the plate onto a rocker in the coldroom.
After 10' pipet off the lysate into a 30 ml corex tube. Add 1/50 vol of 50X PE (0.5 ml for 25 mls; with BME)
Spin tubes in HB-4 for 3' at 3000 rpm at 4 deg.C
Transfer supe to a fresh corex tube and underlay with 2 ml of Ficoll cushion (I use a 6cc syringe with 16 gauge needle with a thin piece of tygon tubing which I can slide to the bottom of the tube).
Spin at 12,700 rpm for 15 at 4 deg.C for 15'. As soon as spin is started, process the next set of plates through steps 4-7.
Aspirate supe till approx. 2 ml above cushion and then collect interface with a pasteur - see Tim's protocol. Collect ~2 ml / tube and pool. Check Ficoll concentration by refractometry and dilute to 10% (w/w) or lower - necessary to make sure it layers onto the sucrose gradient and doesn't sink.
Finish collecting interfaces from second set of plates and then pool all collected interfaces, ensure Ficoll is < 10% (w/w) and load onto 1 gradient. Spin 1 hr - 1hr 30', SW 27 at 2 deg.C.
Fractionate gradient from bottom - 0.3 - 0.5 ml fractions and read sucrose concentration by refractometry. Assay fractions between 48 and 60 % (w/w) sucrose. 5 ul of fraction + 5 ml PE - mix well and pellet onto coverslips: 12,500 rpm for 15' at 4 deg.C in HB-4. Post fix in methanol (-20 deg.C) for 5' and rehydrate and do 5051 + antitubulin followed by anti-mouse, anti-human secondaries. Assess peak by concentration of double staining dots, pool and freeze in liq N2 in 10 ul aliquots.