4. Load 3 mL of the chromoplast lysate on top of the sucrose
density blocks (50%, 40%, 30% and 20% solutions, bottom to
top) prepared in advance in two 13.2 mL polyallomer ultracentrifuge tubes as indicated in Fig. 1b (steps a and b). Afterward,
load the 7% and 0% sucrose gradient blocks on top of the
chromoplast lysate as indicated in Fig. 1b (step c) (see Note 8).
5. Centrifuge at 220,000 Â g for 3 h using a Beckman swinging
bucket rotor SW41Ti (or equivalent), using minimal but
non-null brake settings (see Note 13). A pattern of bands
similar to that shown in Fig. 2c should be obtained (see Note
14).
6. Harvest fractions using a 200 μL micropipette with the tip
trimmed to approximately 1–1.5 mm of diameter doing gentle
circular movements over the surface of the band. Undertake
harvesting sequentially from the upper fraction. The interband
sucrose solutions can be discarded carefully with a 1 mL
micropipette.
7. The isolated fractions can be analyzed immediately or stored at
À20
C or À80
C, depending on the downstream use. Samples for proteomic studies can be stored at À20
C for shortterm usage and at À80
C for longer-term storage. Since
lycopene is very prone to degradation by epoxidation [19]
samples required for carotenoid profiling should be protected
from light, frozen in liquid nitrogen and stored at À80
C for
the shortest time needed. When possible, provide an anoxic
atmosphere inside the tube (nitrogen is suggested) to further
prevent carotenoid breakdown.
4 Notes
1. It is recommended to keep this buffer in constant stirring
before use to ensure that PVPP remains in suspension at the
time of use. This will enhance the effectiveness of polyphenol
removal from the homogenate.
2. Sucrose solutions can alternatively be prepared in the following
way: For 100 mL mix the amount of sucrose needed in each
case (15 g, 30 g 40 g or 50 g) with 10 mL of 0.5 M Tris–HCl
(pH 7.4) buffer and 100 μL of 1 M DTT. Complete to 100 mL
with water and use immediately.
3. Sucrose solutions can be prepared using the alternative method
indicated in Note 2.
4. Extractions from 0.5 to 2.5 kg of starting material have been
successfully performed by scaling up or down the volume of the
isolation buffers and the number and volume of centrifuge
tubes. Chromoplast yield may differ depending on the tomato
variety used.
194
Karel De Pourcq and Albert Boronat
density blocks (50%, 40%, 30% and 20% solutions, bottom to
top) prepared in advance in two 13.2 mL polyallomer ultracentrifuge tubes as indicated in Fig. 1b (steps a and b). Afterward,
load the 7% and 0% sucrose gradient blocks on top of the
chromoplast lysate as indicated in Fig. 1b (step c) (see Note 8).
5. Centrifuge at 220,000 Â g for 3 h using a Beckman swinging
bucket rotor SW41Ti (or equivalent), using minimal but
non-null brake settings (see Note 13). A pattern of bands
similar to that shown in Fig. 2c should be obtained (see Note
14).
6. Harvest fractions using a 200 μL micropipette with the tip
trimmed to approximately 1–1.5 mm of diameter doing gentle
circular movements over the surface of the band. Undertake
harvesting sequentially from the upper fraction. The interband
sucrose solutions can be discarded carefully with a 1 mL
micropipette.
7. The isolated fractions can be analyzed immediately or stored at
À20
C or À80
C, depending on the downstream use. Samples for proteomic studies can be stored at À20
C for shortterm usage and at À80
C for longer-term storage. Since
lycopene is very prone to degradation by epoxidation [19]
samples required for carotenoid profiling should be protected
from light, frozen in liquid nitrogen and stored at À80
C for
the shortest time needed. When possible, provide an anoxic
atmosphere inside the tube (nitrogen is suggested) to further
prevent carotenoid breakdown.
4 Notes
1. It is recommended to keep this buffer in constant stirring
before use to ensure that PVPP remains in suspension at the
time of use. This will enhance the effectiveness of polyphenol
removal from the homogenate.
2. Sucrose solutions can alternatively be prepared in the following
way: For 100 mL mix the amount of sucrose needed in each
case (15 g, 30 g 40 g or 50 g) with 10 mL of 0.5 M Tris–HCl
(pH 7.4) buffer and 100 μL of 1 M DTT. Complete to 100 mL
with water and use immediately.
3. Sucrose solutions can be prepared using the alternative method
indicated in Note 2.
4. Extractions from 0.5 to 2.5 kg of starting material have been
successfully performed by scaling up or down the volume of the
isolation buffers and the number and volume of centrifuge
tubes. Chromoplast yield may differ depending on the tomato
variety used.
194
Karel De Pourcq and Albert Boronat