This protocol is derived from (Lundborg et al., 1981; Widell and Larsson, 1981) with minor modification, it was optimized for Solanum chacoense which contains a lot of phenolics. PVPP and PVP may be added in smaller amount when using Arabidopsis.
Amount of starting material can be scaled down according to protein abundance and needs. It is recommended to start with green material, it make phases separation much easier to visualize.
All steps must be carried out at 4ºC or on ice, buffers are kept at 4ºC.
0,5 M | sucrose |
50 mM | HEPES-KOH pH 7,5 |
5 mM | dithioerythritol |
0,6% (w/v) polyvinyl pyrrolidone | |
1% polyvinyl polypyrrolidone. Add 12 hour before use and agitate at 4ºC overnight | |
1% β-mercaptoethanol | |
1X protease cocktail inhibitor (Roche). Add just prior to use |
0.33 M | sucrose |
3 mM | KCl |
5 mM | potassium phosphate pH 7,8 |
Phase mixture and phase system are prepared 24 hours before use. On the morning of the fractionation the phase system (which has separated overnight) is separated between upper and lower phase and kept at 4ºC.
Step | Phase mixture | Phase system |
---|---|---|
20% (W/W) Dextran T-500 | 11.16 g | 93g |
5.58g | 46.50g | |
Sucrose solid | 3.05g | 33.89g |
0.2M Potassium phosphate pH 7,8 | 0.675 mL | 7.5 mL |
2M KCl | 0.041 mL | 0.45 mL |
Add ddi Water to a final weight of | 27g | 300 |
For plant material