hi,
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?
Centrosome Isolation
( 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
Lysis Buffer:
1 mM Hepes (pH 7.2),
0.5% Nonidet P-40,
0.5 mM MgCl2,
0.1% 2-mercaptoethanol
proteinase inhibitors
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).
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