Hello all,
I have recently started working with cytosolic/nuclear fractioning of HEK293T cells. My first few attempts were unsuccessful, in that there seemed to be pretty much equal contamination of cytosolic material in the nuclear samples, and vice versa. This determination was made based on the presence of nuclear controls in the cytosol and cytosolic controls in the nuclear samples.
With repeated rounds of optimization, we landed on what seems to be an at least passable method wherein nuclear controls are MOSTLY showing up in the nucleus and so forth.
The problem I'm now running into is this - my cytosolic control (Tubulin) is showing up mostly in the cytosol (as expected), and the same with Histone H3 (a nuclear control). However, a second nuclear control, PCNA, is showing up more in the cytosol, which does not make sense. It's strange to me that the other two controls seem to be showing expected results, but not PCNA.
Has anyone ever run into problems like this? If so, how did you resolve them?
Thank you!
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