Protocols > Nuclear fraction

Preparation of cytosolic and nuclear protein fractions.

Procedure

  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.

Solutions required



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




Courtesy of Dr. Laszlo Bako, Umeå Plant Science Centre

For other methods, please check: Plant Methods (Open access)

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