Centrosomes are present in the cytosolic fraction? - (Apr/14/2010 )

Hi everybody,
Please correct me if I am wrong in my protocol of subcellular fractionation or my suppositions.
My protocol of centrifugation is:

Immediately after cell lysis, centrifugation 10 min x1000g at 4ºC (this pellet corresponds to nuclei, debris and mitochondria).
Of the corresponding supernatant, I perform ultracentrifugation: 1 hour x100000g at 4ºC
The pellet corresponds to membrane fraction and the supernatant is the cytosolic fraction.
I am using the cytosolic fraction for IP and LC-MS.

My question is...
I suspect my protein of interest is centrosomal or has something to do with centrosomal proteins.
With my simple protocol of ultracentrifugation centrosomes are included in the cytosolic fraction, aren't they?
I mean...I am not losing any centrosomal protein after ultracentrifugation following this protocol (the protocol to isolate centrosomes is much more complicated, so I suppose I would need a gradient centrifgation with specific solutions...).
I'm wrong?

The centrosomes should be in the cytosolic fraction. However, I suspect for your protocol that detection of the centrosome will be difficult because of dilution during the fractionation.

bob1 on Apr 14 2010, 05:05 PM said:

Thank you for your answer, but...what do you mean with "dilution"?

The cells in their intact state have a cellular volume, of which the centrosome is a small part. When you lyse the cells you are diluting the cellular components out. To remove intact nuclei, you will have used a largish volume of a fairly weak salt solution - which now contains your centrosomes. Presumably to detect the centrosome you will need to run it on a gel of some sort... the diluted lysed cells may mean that there might not be enough centrosome in the volume that you can load on a gel, for you to detect.

You loose the centrosomes in the first centrifugation. Centrosomes are highly associated with the nucleus and spin down with them. You must treat cells with nocodazole and cytochalasin D first to dissociate centrosomes. I highly suggest you do IF studies to look at localization!! My entire thesis work revolved around centrosomes and I've done battle with reviewers over this point. Plus, if your localization is cell cycle dependent, it will be next to impossible to get enough protein for western blot in an asynchronous population.

rkay447 on Apr 16 2010, 01:16 AM said:

In fact, I suspect my protein could be centrosomal or related with centrosomes after localization studies of my protein fusioned with GFP; it looks like a centrosomal pattern. In my immunoprecipitation studies I use an anti-GFP antibody and I detect my fusion protein in the citosolic protein, as well as some candidate proteins that could be interacting with muy protein of interest. I am concerned about I could be losing some other good candidates because of my centrifugation protocol (I know it's necessary to do IF with gamma-tubulin for example to verify if my protein is really located in centrosomes, and perhaps to purify centrosomes if my hypothesis is validated).