Isolation of the nuclear fraction

The protocol for nuclear fractionation

1. About 0.5-1.0g young seedlings were ground to a fine powder with liquid nitrogen.
2. Add an equal volume of protein lysis buffer and lyse on ice for 10-15 minutes. Protein Lysis Buffer (20 mM Tris-HCl [pH 7.5]，20 mM KCl，2 mM EDTA，2.5 mM MgCl₂，250 mM Sucrose, 5 mM DTT, 25% glycerol and protease inhibitor cocktail).
3. The solution was filtered through two layers of Miracloth (Calbiochem, San Diego, USA) to remove plant debris. Take 100 μL of the filtrate as total protein and place it on ice. 
4. The obtained solution was centrifuged at 1500 x g for 10 min at 4°C. The filtrate was divided into pellet and supernatant. Take 100 μL of the supernatant as the non-nuclear protein and place it on ice.
5. Wash the pellet with 600 μL of re-suspension buffer (NRBT: 20 mM Tris-HCl [pH 7.5], 2.5 mM MgCl₂, 0.2% Triton X-100, 25% glycerol and protease inhibitor cocktail) for a total of five times at 4°C, 1500 g, and centrifuge for 10 min. 
6. After washing, the pellet was suspended in 300 μL NRB2 (20 mM Tris-HCl [pH 7.5], 250 mM sucrose, 10 mM MgCl₂, 0.5% Triton X-100, 5 mM β-mercaptoethanol and protease inhibitor cocktail).
7. Slowly added on top of the same volume of NRB3 (20 mM Tris-HCl [pH 7.5], 1.7 M sucrose, 10 mM MgCl₂, 0.5% Triton X-100, 5 mM β-mercaptoethanol and protease inhibitor cocktail). 
8. After centrifugation at 16,000 x g for 45 min at 4°C, the pellet was suspended in lysis buffer as the nuclear fraction.
9. The total protein, non-nuclear protein, and nuclear protein were loaded respectively, and the distribution of target protein was detected by western blotting.