Hi Yvonne, Nuclei usually pellet in the first spin ~1000g, along with large sheets of plasma membrane, unbroken cells, and debris. To get a pure nuclear fraction, people usually use sucrose gradients or some other method to separate the whole nuclei from the other junk. To get a pure cytosol fraction, you should do a 100,000g spin (at least 1hr at 4C).
I don't have a protocol for a nuclear extraction, but a general framework would look something like this: 1. Dissect out brain region(s) of interest. Mince into small pieces. Throughout the protocol, all work should be on ice and all spins should be at 4C. 2. Add buffer, usually 1 vol of something like PBS (1 volume equivalent means 100ul for 100mg tissue, etc). Homogenize. 3. Spin at 1000g for 10-15min to pellet nuclei, unbroken cells, plasma membrane, and debris. The supernatant will be a crude cytosolic prep including small membrane-bound organelles. 4a. Supernatant from 3: spin at 100,000g for 1hr to pellet everything that's not soluble, leaving a pure cytosolic prep as your supernatant. 3b. Pellet from 3: additional spins/gradients to separate nuclei from other stuff. Your buffer will need to balance salts, etc, in order to preserve the nuclei intact until you have a pure nuclear fraction. Hope this helps, Irit On Oct 13, 2011, at 6:53 AM, Yvonne Couch wrote: > Hi all, > I'm looking for a basic protocol for nuclear and cytosolic extractions from > small (c.50mg) amounts of brain tissue. I have a kit from WebScientific which > doesn't appear to have enough centrifugation steps and also calls for very > little tissue homogenization prior to spinning so I feel like all I am doing > is breaking the tissue into small lumps and spinning it down without actually > breaking it open to get the cytoplasm out. > > Does anyone have a nice nuclear/cytosolic protocol with home made buffers and > any ideas as to what the solution/tissue should look like at each stage? > Thanks > Yvonne > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
