4. Centrifuge the supernatant from step 3 at 27,000 Â g, 4
C for
1 h.
5. Decant the supernatant (cytosol and membrane) into clean
50 mL Oakridge centrifuge tubes.
6. Resuspend the pellet (cell wall) in breaking buffer without
DNase or RNase (30 mL) and form a homogeneous suspension by vortexing.
7. Centrifuge the cytosol and membrane suspension from step 5
and the resuspended cell wall pellet from step 6 at 27,000 Â g,
4
C for 1 h to remove residual contaminating material.
8. From the resuspended cell wall pellet centrifuged in step 7,
decant and discard the supernatant then store the cell wall
pellet at 4
C.
9. From the cytosol and membrane sample centrifuged in step 7,
collect the supernatant (clarified cytosol and membrane) in
40 mL ultracentrifuge tubes and discard the pellet.
10. Balance the ultracentrifuge tubes very precisely (see Note 66).
11. Centrifuge the clarified cytosol and membrane at 100,000 Â g,
4
C for 4 h.
12. Collect the supernatant (cytosol) in clean 40 mL ultracentrifuge tubes and reserve the pellet (membrane).
13. Gently wash the membrane pellets in breaking buffer without
DNase or RNase and store at 4
C (see Note 67).
14. Repeat centrifugation of cytosol as in steps 10 and 11 (Fig. 6).
15. Collect the final 100,000 Â g supernatant (cytosol) in 50 mL
conical tubes and wash the membrane pellets as in step 13.
16. Resuspend the reserved cell wall pellet(s) from step 8 in
10 mM ammonium bicarbonate and pool cell wall pellets
together.
17. Resuspend the membrane pellets from steps 12 and 15 in
10 mM ammonium bicarbonate and pool.
18. Cut lengths of dialysis tubing long enough to fit the volumes of
each product, being sure to allow room for expansion during
dialysis. Rehydrate dialysis tubing in water for 30 min (or according to manufacturer’s recommendation).
19. Secure one end of the dialysis tube, either by tying or by
attaching a dialysis clip, fill with sample, and secure the other
end (see Note 68).
20. Place the filled dialysis tubes in 7 L of 10 mM ammonium
bicarbonate and place at 4
C for 4–16 h.
21. Change to fresh 10 mM ammonium bicarbonate and dialyze
for another 4–16 h.
22. Repeat once more for a total of three buffer exchanges.
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Megan Lucas et al.
C for
1 h.
5. Decant the supernatant (cytosol and membrane) into clean
50 mL Oakridge centrifuge tubes.
6. Resuspend the pellet (cell wall) in breaking buffer without
DNase or RNase (30 mL) and form a homogeneous suspension by vortexing.
7. Centrifuge the cytosol and membrane suspension from step 5
and the resuspended cell wall pellet from step 6 at 27,000 Â g,
4
C for 1 h to remove residual contaminating material.
8. From the resuspended cell wall pellet centrifuged in step 7,
decant and discard the supernatant then store the cell wall
pellet at 4
C.
9. From the cytosol and membrane sample centrifuged in step 7,
collect the supernatant (clarified cytosol and membrane) in
40 mL ultracentrifuge tubes and discard the pellet.
10. Balance the ultracentrifuge tubes very precisely (see Note 66).
11. Centrifuge the clarified cytosol and membrane at 100,000 Â g,
4
C for 4 h.
12. Collect the supernatant (cytosol) in clean 40 mL ultracentrifuge tubes and reserve the pellet (membrane).
13. Gently wash the membrane pellets in breaking buffer without
DNase or RNase and store at 4
C (see Note 67).
14. Repeat centrifugation of cytosol as in steps 10 and 11 (Fig. 6).
15. Collect the final 100,000 Â g supernatant (cytosol) in 50 mL
conical tubes and wash the membrane pellets as in step 13.
16. Resuspend the reserved cell wall pellet(s) from step 8 in
10 mM ammonium bicarbonate and pool cell wall pellets
together.
17. Resuspend the membrane pellets from steps 12 and 15 in
10 mM ammonium bicarbonate and pool.
18. Cut lengths of dialysis tubing long enough to fit the volumes of
each product, being sure to allow room for expansion during
dialysis. Rehydrate dialysis tubing in water for 30 min (or according to manufacturer’s recommendation).
19. Secure one end of the dialysis tube, either by tying or by
attaching a dialysis clip, fill with sample, and secure the other
end (see Note 68).
20. Place the filled dialysis tubes in 7 L of 10 mM ammonium
bicarbonate and place at 4
C for 4–16 h.
21. Change to fresh 10 mM ammonium bicarbonate and dialyze
for another 4–16 h.
22. Repeat once more for a total of three buffer exchanges.
94
Megan Lucas et al.