MEMBRANE SEPARATIONS
 
Last Update: December 2006
 
 
PROCEDURE - Differential Centrifugation
1. Centrifuge total protoplast protein (see protocol 128, Protoplast preparation from plant tissues) at 1,000 xg for 20 min at 4°C
2. Wash the pellet (p1) with lysis buffer and centrifuge again. Centrifuge the supernatant (s1) at 5,000 xg for 30 min
3. Wash the pellet with lysis buffer and centrifuge again (now called p5). Treat the supernatant (now s5) in the same way for sequential differential centrifugations at 15,000, 25,000, 50,000, and 100,000 xg, with the last two centrifugations carried out for 40 min

4. Resuspended all pellets (p1, p5, p15, p25, p50, and p100) in lysis buffer and quantify the protein
5. The protein samples can now be analyzed by Western blot
 
PROCEDURE - Membrane Association
1. Prepare total microsomes by centrifugation of total protoplast protein (see protocol 128) at 150,000 xg for 1 h and washing the pellet with lysis buffer
2. Resuspended pellets with lysis buffer (total protein) or lysis buffer containing 0.1, 0.5, or 1.0% Triton X-100, 0.5 or 2 m urea, 0.1M sodium carbonate, 0.1, 0.5, or 1.0 M NaCl, or 0.1M potassium phosphate, pH 7.0, for 1 h on ice, and centrifuged again at 150,000 xg for 1 h
3. Wash the resulting pellets with lysis buffer, resuspend in loading buffer, and separated by SDS-PAGE prior to analysis by Western blot using antibodies
 
PROCEDURE - Succrose-Density-Gradient Centrifugation
1. Centrifuge total protoplast protein at 1000 xg for 10 min at 4°C
2. Load 6 mL of the supernatant (S1) on top of a 16 to 55% equilibrium-density sucrose gradient modified from: DM Gibeaut and NC Carpita (1994) Biosynthesis of plant cell wall polysaccharides. The FASEB Journal, Vol 8: 904-915
3. Prepare the gradient using stock solutions of 55, 40, 33.5, 26.5, and 16% (w/v) sucrose buffered in 10mM Hepes-KOH, pH 7.0, 10mM potassium acetate, and 2mM EDTA

4. Prepare gradients by the sequential addition of 2.7 mL of 55% sucrose solution, 8.8 mL of 40% sucrose solution, 7.0 mL of 33.5% sucrose solution, 6.0 mL of 26.5% sucrose solution, and 4.5 mL of 16% sucrose solution to 35 mL polyallomer tubes (catalog no. 326823, Beckman)
5. After carefully layering these solutions, centrifuge at 150,000 xg for 18 to 20 h at 4°C in a rotor (model SW28, Beckman)
6. At the end of the centrifugatioin, an equilibrium-density gradient similar to one obtained using a gradient maker should be obtained
7. Collect equal-volume fractions from the bottom of the gradients. Estimate the sucrose concentration of each fraction by measuring the refractive index using a refractometer
8. Separate aliquots of the fractions (50-100 µL) by SDS-PAGE, and analyze the presence of specific proteins, and membrane marker proteins, by Western blot