Antibodies Plant/Algal / DNA/RNA/Cell Cycle / Transcription regulation

Preparation of cytosolic and nuclear protein fractions

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.
GH buffer: 100mM glycine
0.1% Hexylene glycol
0.37M (4.7% w/v) saccharose
0.3mM Spermine
1.0mM Spermidine
pH 8.3 with Ca(OH)₂
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 5min sediment nuclei by centrifugation at 1000 xg for 15 min at 4°C. Save supernatant as the
cytoplasmic fraction. Wash the pelleted nuclei two times with GHT (GH+0.1% TX100) then finally
resuspended in a suitable volume of extraction buffer + protease inhibitors.

Courtesy Dr. Laszlo Bako, Umeå Plant Science Centre