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.
Extraction buffer (prepare 200 ml)
HEPES-KOH pH 7,5
0,6% (w/v) polyvinyl pyrrolidone
1% polyvinyl polypyrrolidone. Add 12 hour before use and agitate at 4ºC overnight
1X protease cocktail inhibitor (Roche). Add just prior to use
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.
20% (W/W) Dextran T-500
0.2M Potassium phosphate pH 7,8
Add ddi Water to a final weight of
Fifty grams of plant leaves were homogenized using a pre-chilled kitchen blender in extraction buffer. Try to keep volume minimal, 75 ml should do.
The homogenate was filtered twice through four layers of cheese cloth and spun at 10 000 X g for 10 minutes.
The supernatant was transferred to a clean ultracentrifuge tube (Beckman) and spun at 50 000 X g for 1h.
The microsomal pellet was resuspended in 10 mL resuspension buffer.
9g of microsomal pellet was added to 27 g of phase mixture.
A total of five passes were done on fresh phase systems. Upper phase was mixed with fresh lower phase and lower phase was mixed with fresh upper phase.
The fractions were diluted with the resuspension buffer; 2-fold for the plasma membrane fraction and 10-fold for the fraction corresponding to the intracellular membranes and were spun at 100,000g for 1h.
The purified membrane pellet were resuspended in 0,1 M Tris-HCl pH 7,5, SDS 0,1%, 2% β-mercaptoethanol, and 1X protease cocktail inhibitor.
Concentration was verified using a Bradford protein assay (Bio-RAD) and ten mg was mixed with Laemli buffer and heated to 90ºC for 5 minutes and stored at -20ºC until use.