When you carry out lysis of mammalian cells with a "regular" lysis buffer such as phosphate buffer, 150 mM NaCl, and detergent such as 1% Triton X-100, the centrosomes stay attached to the nucleus. So, if you lyse the cells and pellet the nuclei (do not sonicate) by centrifuging for 10,000-13,000 x g for 20-30 minutes, the centrosomes will be in the nuclear pellet.
You can check this by looking at your cytosolic supernatent and the nuclear pellet using immunofluoresence to detect a couple of centrosome proteins. I have done this before with 1% Triton X-100 as a detergent and also using 0.2% CHAPS as the detergent. I observed centrosomes attached to almost all of the nuclei in the pellet. Of course you will still have centrosome proteins in the cytosol, since many centrosomes are present both in the cytosol and the centrosome (depending on the protein). i did this with HeLa cells and also with U2OS.
Hope this helps,
Nicola
When you carry out lysis of mammalian cells with a "regular" lysis buffer such as phosphate buffer, 150 mM NaCl, and detergent such as 1% Triton X-100, the centrosomes stay attached to the nucleus. So, if you lyse the cells and pellet the nuclei (do not sonicate) by centrifuging for 10,000-13,000 x g for 20-30 minutes, the centrosomes will be in the nuclear pellet.
You can check this by looking at your cytosolic supernatent and the nuclear pellet using immunofluoresence to detect a couple of centrosome proteins. I have done this before with 1% Triton X-100 as a detergent and also using 0.2% CHAPS as the detergent. I observed centrosomes attached to almost all of the nuclei in the pellet. Of course you will still have centrosome proteins in the cytosol, since many centrosomes are present both in the cytosol and the centrosome (depending on the protein). i did this with HeLa cells and also with U2OS.
Hope this helps,
Nicola
Interesting question! Most buffers with some combination of salts and detergents will solubilize organelles to some extent. Differential centrifugation (rather than specific buffers) maybe best. You could check papers wherein the mitotic spindle was isolated. Maybe you can first remove the spindle and centrosomes with these buffers/methods...the rest is cytoplasmic material (which you want). Hope this helps!
You can try using digitonin, which permabilizes the plasma membrane, but not the nuclear one.
http://jcb.rupress.org/content/111/3/807.full.pdf
I am looking for an answer as well, but I want to the centrosomes to Co-IP. Any help?
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