Hi Cheri, I am not quite sure why you will need to use an ultracentrifuge to separate cytoplasmic and nuclear fractions of cell extracts? Usually, you can do that by usage of different buffers for the cytoplasmic fraction and the nuclear fraction.

Thank you for responding. I agree and I am currently using buffers based on the Schreiber method. I am using anti-retinoblastoma (Rb) by western blot to check the quality of the fractions. Rb is showing up in my cytoplasmic fraction. I was advised that I need ultracentrifugation to pellet nuclei and that is why I have contamination of the cytoplasmic fraction with a nuclear protein. I am reluctant to believe that and so am wondering why I would want to use ultracentrifugation on the nuclei. What do you think?

I am using low speed (3,500 rpm) for getting the cytoplasmic fraction. Afterwards, I included a washing step with the same buffer to remove really all cytoplasmic fraction and spin a bit higher (4,000 rpm). Then I am going to either resuspend my nuclear fraction in Laemmli buffer in case I just need it for direct western or perform an extraction of nuclear soluble fraction to perform am immunoprecipitation.

I had never thought of resuspending the nuclear pellet with Laemmli buffer. Thank you very much for your comments!

You are very welcome. In case you just need your fraction for direct western you can resuspend it in 1x Laemmli buffer, boil it (probably a bit longer than 5 min) and load equal volumes relative to the cytoplasmic fraction on your gel (no Bradford measurement possible). Good luck!