Nuclear Fraction Services

1. Prepare protoplasts from 50 ml Arabidopsis thaliana cell culture according to the protocol of PEG transfection.
2. Resuspend protoplasts in 10 ml GH buffer and keep the solution on ice for 10 min.
3. To release nuclei, add Triton X100 to a final concentration of 0.1%. Pipetting gently up and down several
   times with a plastic pipette might be necessary to lyse cells.
4. After 5 min, sediment nuclei by centrifugation at 1000 xg for 15 min at 4°C.
5. Save supernatant as the cytoplasmic fraction.
6. Wash the pelleted nuclei two times with GHT (GH+0.1% TX100).
7. Resuspended the pellet in a suitable volume of extraction buffer with protease inhibitors.

GH BUFFER

• Glycine : 100 mM
• Hexylene glycol : 0.1%
• Saccharose : 0.37M (4.7% w/v)
• Spermine : 0.3 mM
• Spermidine : 1.0 mM
• pH 8.3 with Ca(OH)2