5. Discard the supernatant and gently resuspend each pellet in
3 mL of buffer B using a 1 mL micropipette with the tip
trimmed to approximately 2–3 mm of diameter to reduce
chromoplast damage during handling (see Note 7).
6. Pool the resuspended pellets into a 30 mL polypropylene centrifuge tube using a 1 mL micropipette with the tip opening
trimmed to approximately 2–3 mm of diameter. Complete to
25 mL with buffer B.
7. Centrifuge at 5000 Â g for 10 min in rotors JA-17, JA-25.50,
or similar.
8. Carefully discard the supernatant by decantation and gently
resuspend the chromoplasts in 4 mL of buffer B using a 1 mL
micropipette with the tip trimmed to approximately 2–3 mm of
diameter.
9. Carefully load the 4 mL chromoplast sample on top of the
sucrose density blocks (50%, 40%, 30% and 15%—bottom to
top) prepared in advance in a 38.5 mL polyallomer ultracentrifuge tube as indicated in Fig. 1a (see Note 8). Add buffer B as
required to equilibrate the tubes before ultracentrifugation.
10. Centrifuge at 100,000 Â g for 60 min in a Beckman swinging
bucket SW 28 Ti rotor (or equivalent) using mild brake
settings.
11. Carefully withdraw the chromoplasts present in the 30–40%
interface (Fig. 2a) using a wide-bore plastic Pasteur pipette and
transfer into a polypropylene 30 mL centrifuge tube (see Note
9).
12. Add one volume of buffer B and centrifuge at 5000 Â g for
10 min.
13. Carefully remove the supernatant by decantation and remove
the remaining buffer droplets using a 200 μL micropipette (see
Note 10).
14. Continue with the chromoplast fractionation steps as indicated
in Subheading 3.2.
3.2 Chromoplast
Fractionation
1. Resuspend the pellet in 3 mL of lysis buffer using a 1 mL
micropipette and complete the final volume to 6 mL. Add
PMSF to 1 mM final concentration and mix with vigorous
agitation (see Note 11).
2. Lyse chromoplasts by sonication keeping the tube in an
ice-filled beaker. Three 30 s pulses with pauses of at least
15–20 s are recommended (see Note 12).
3. Remove remaining unbroken chromoplasts and debris by centrifugation at 5000 Â g for 10 min.
Chromoplast Fractionation
193
3 mL of buffer B using a 1 mL micropipette with the tip
trimmed to approximately 2–3 mm of diameter to reduce
chromoplast damage during handling (see Note 7).
6. Pool the resuspended pellets into a 30 mL polypropylene centrifuge tube using a 1 mL micropipette with the tip opening
trimmed to approximately 2–3 mm of diameter. Complete to
25 mL with buffer B.
7. Centrifuge at 5000 Â g for 10 min in rotors JA-17, JA-25.50,
or similar.
8. Carefully discard the supernatant by decantation and gently
resuspend the chromoplasts in 4 mL of buffer B using a 1 mL
micropipette with the tip trimmed to approximately 2–3 mm of
diameter.
9. Carefully load the 4 mL chromoplast sample on top of the
sucrose density blocks (50%, 40%, 30% and 15%—bottom to
top) prepared in advance in a 38.5 mL polyallomer ultracentrifuge tube as indicated in Fig. 1a (see Note 8). Add buffer B as
required to equilibrate the tubes before ultracentrifugation.
10. Centrifuge at 100,000 Â g for 60 min in a Beckman swinging
bucket SW 28 Ti rotor (or equivalent) using mild brake
settings.
11. Carefully withdraw the chromoplasts present in the 30–40%
interface (Fig. 2a) using a wide-bore plastic Pasteur pipette and
transfer into a polypropylene 30 mL centrifuge tube (see Note
9).
12. Add one volume of buffer B and centrifuge at 5000 Â g for
10 min.
13. Carefully remove the supernatant by decantation and remove
the remaining buffer droplets using a 200 μL micropipette (see
Note 10).
14. Continue with the chromoplast fractionation steps as indicated
in Subheading 3.2.
3.2 Chromoplast
Fractionation
1. Resuspend the pellet in 3 mL of lysis buffer using a 1 mL
micropipette and complete the final volume to 6 mL. Add
PMSF to 1 mM final concentration and mix with vigorous
agitation (see Note 11).
2. Lyse chromoplasts by sonication keeping the tube in an
ice-filled beaker. Three 30 s pulses with pauses of at least
15–20 s are recommended (see Note 12).
3. Remove remaining unbroken chromoplasts and debris by centrifugation at 5000 Â g for 10 min.
Chromoplast Fractionation
193