We are trying to isolate yeast nuclei for footprinting purposes, since on bandshifts we get a fairly weak complex using crude extracts.
We have encountered a number of problems:
1. The spheroplasts wouldn't lyse in either 18% Ficoll 400 or 8% PVP 40, even after extensive homogenisation with a Polytron. They are real spheros, since they lyse in water
and have no cell wall under the microscope.
2. We use the Percoll gradient method, but nearly everything bands in the middle where one would expect the nuclei, but under the microscope
they look like spheroplasts. DAPI staining shows a strong signal inside, but there are still lots of "cytoplasm" around. They are also the same size
as the sheroplasts.
3. We have followed cytoplasmic lysis with ADH activity, but the majority is still associated with our "nuclei".
4. Are they really so hard to lyse?