Help! Nuclear extraction! - (Jul/14/2006 )
I'm trying to extract nuclei from rat macrophages. Briefly, after lysis of plasma membrane and collect the pellet (containing nuclei), I washed 3 times with lysis Buffer and then broke down the nuclear membrane. However, my nuclear extraction always contaimed with cytosolic proteins (detecting via Western Blot with antibody against a-tubulin). I wonder if there is other method to minimize the cytosolic contamination in the nuclear extraction.
I dont think any subcellular fraction is really pure. Its always contaminated with some other fractions, atleast a little. I used to do mitochondrial fractionation and always found some mitochondria in the nuclear fractionation. Experience will decrease contamination of other fractions.
mayb some one sells a kit which might give quite a pure nuclear fraction. But have to search for it.
I suggest that you homogenize your cell lysate with a glass Dounce homoginizer for at lease 10 times, then centrifuge it through a 15-30% sucrose cushion layer. This should help.
in that topic i've posted the protocols for separation of proteins from cyto and nucleus.
The rolling time may be adjusted. 30' is routine and may be adjusted to 1h or more