I am separating cellular fractions of liver tissues for western blot. So far I have been successful with my cytosolic and mitochondrial fractions but struggling to increase the purity of my nuclear fraction. My nuclear lysis buffer is comprised of: 20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5 M NaCl, 0.2 mM EDTA, 20% glycerol, 1% Triton-X-100, protease, and phosphatase inhibitors.

I washed the pellet with my lysis buffer which I also use for cytosolic extraction: 20mM Mannitol, 50 mM sucrose, 1mM EDTA, 0.5 mM EGTA (pH 7.4), 10 mM Tris-HCl (pH 6.8). 5mM NAD, 5uM Trichostatin A, plus protease and phosphatase inhibitors. Centrifuged at 800g for 15 minutes and did this 3x then increased to 5x to wash off cell debris (still has contamination). After which I resuspended the pellet with my nuclear lysis buffer, kept it on ice for 30 mins, and sonicated for 10s 3x with 30s intervals, then centrifuged for 9000g for 30 mins (supernatant was my nuclear fraction).

My question is, how do I increase the purity of my nuclear fraction? Thank you for your insights.