I'm doing some preliminary experiments related to the traslocation of a protein to the nucleus under stress conditions, but I've never done the separation of the nuclei from the cytoplasm. I need a quick protocol, which possibly considers the use of "standard" reagents available in every cell biology/biochemsitry lab.
Hi Nicoletta,
The most common way to purify nuceli from cultured cells is to use hypotonic buffer and dounce homogenizer. This will allow you to lyse the cells while keeping the nuclei intact, thus separating the cytoplasmic fraction from the nucleoplasmic fraction after centrifugation and sucrose cushion separation.
You can find a good detailed protocol here :
http://www.lamondlab.com/f7nucleolarprotocol.htm
Good luck with your experiments :)
Hi Nicoletta,
The most common way to purify nuceli from cultured cells is to use hypotonic buffer and dounce homogenizer. This will allow you to lyse the cells while keeping the nuclei intact, thus separating the cytoplasmic fraction from the nucleoplasmic fraction after centrifugation and sucrose cushion separation.
You can find a good detailed protocol here :
http://www.lamondlab.com/f7nucleolarprotocol.htm
Good luck with your experiments :)
Thank you very much for the quick answer. I had a look at the protocol you suggested, but at the moment we don't have a tissue homogenizer... Finding a homogenizer will require some time: can you suggest me a way to substitute that step in the protocol with something else doable without the homogenizer?
Another way, which might be not as clean as the one decsribed above, is to use detergent and centrifugation. It requires less specific material.
Have a look a this protocol, and tell me if it is more suitable for your lab :
http://faculty.virginia.edu/mirmira/resources_files/Protocols/Nuclear%20Extracts.htm
Thanks! This one seems much easier and we can easily have all the needed reagents. In the meantime, I will look for the tissue homogenizer, since you said that the first protocol leads to a better separation and cleaner material.
I'll let you know! :-)
Hi Nicoletta,
Please let me know whether the protocol provided by Nicolas yielded good results for you.
Thanks
Is there any other better protocol. I work with mES cells and not sure if using a homogeniser will still help in retaining the property well or not. Please help.
Here is described fairly simple method how to separate nucleoplasm, nuclear membranes and cytoplasm without a need of homogenizer:
Article Subcellular Fractionation
Also, one may find good cellular fractionation protocols in these papers (see attached)
- Badges
- Science method