5. A typical extraction starting from 1 kg of fruits can yield around
600–700 g of tissue, which will entail the use of about
1.2–1.4 L of buffer A.
6. For preparations starting from large amounts of fruit a set of
several filter funnels can be used in parallel to minimize the
handling time.
7. Note that the upper layer of the pellets can be readily resuspended, while the lower part is more compact. Full resuspension can be facilitated by covering the remnant of the pellets
with a small volume of buffer B (about 3 mL) and waiting for a
few minutes before continuing. Carry on with this procedure
until all pellets are resuspended.
8. Loading of the sucrose density blocks can be done in two ways:
(a) using a fine-bore glass Pasteur pipette or (b) using a peristaltic pump coupled to a capillary tube. The exact volume of
each sucrose block can be dispensed in advance into separate
assay tubes. (a) If using a Pasteur pipette, very gently let flow a
narrow stream of the sucrose solution on top of the previous
(denser) block. Dispense the solutions against the tube wall
from a close distance to the surface. Start with the 50% sucrose
block and continue in an ascending way to the 40, 30, and 15%
sucrose blocks. (b) If using a peristaltic pump, the gradient is
prepared in an inverse way. The pump tubes are connected to a
long capillary tube that has to be placed in contact with the
bottom of the centrifuge tube. The pump will be fed sequentially with the right volumes of the 15%, 30%, 40%, and 50%
sucrose solutions. The gradient blocks will be thus built from
the bottom to the top. When removing the capillary tube, do it
slowly and ensuring that no air bubbles leave from the capillary
(which would disturb the gradient).
9. This fraction contains fully developed chromoplasts [17]. To
minimize cross-contamination it is strongly recommended to
withdraw the fractions present at the 0–15% and 15–30% interfaces in advance. Harvest the chromoplasts while gently
moving the tip over the surface of the band. This makes the
harvesting more effective (otherwise, inter-band material can
easily be taken up).
10. The chromoplast pellet is very unstable at this particular stage.
To minimize chromoplast losses perform decantation of the
supernatant immediately after removing the tube from the
centrifuge by carefully pouring the supernatant in the direction
opposed to the pellet.
11. Chromoplast resuspension is more effective if initially done
into a smaller volume before completing to the full volume.
This forces any chromoplast clumps to pass more frequently
through the pipette tip opening, favoring their disaggregation.
Chromoplast Fractionation
195
600–700 g of tissue, which will entail the use of about
1.2–1.4 L of buffer A.
6. For preparations starting from large amounts of fruit a set of
several filter funnels can be used in parallel to minimize the
handling time.
7. Note that the upper layer of the pellets can be readily resuspended, while the lower part is more compact. Full resuspension can be facilitated by covering the remnant of the pellets
with a small volume of buffer B (about 3 mL) and waiting for a
few minutes before continuing. Carry on with this procedure
until all pellets are resuspended.
8. Loading of the sucrose density blocks can be done in two ways:
(a) using a fine-bore glass Pasteur pipette or (b) using a peristaltic pump coupled to a capillary tube. The exact volume of
each sucrose block can be dispensed in advance into separate
assay tubes. (a) If using a Pasteur pipette, very gently let flow a
narrow stream of the sucrose solution on top of the previous
(denser) block. Dispense the solutions against the tube wall
from a close distance to the surface. Start with the 50% sucrose
block and continue in an ascending way to the 40, 30, and 15%
sucrose blocks. (b) If using a peristaltic pump, the gradient is
prepared in an inverse way. The pump tubes are connected to a
long capillary tube that has to be placed in contact with the
bottom of the centrifuge tube. The pump will be fed sequentially with the right volumes of the 15%, 30%, 40%, and 50%
sucrose solutions. The gradient blocks will be thus built from
the bottom to the top. When removing the capillary tube, do it
slowly and ensuring that no air bubbles leave from the capillary
(which would disturb the gradient).
9. This fraction contains fully developed chromoplasts [17]. To
minimize cross-contamination it is strongly recommended to
withdraw the fractions present at the 0–15% and 15–30% interfaces in advance. Harvest the chromoplasts while gently
moving the tip over the surface of the band. This makes the
harvesting more effective (otherwise, inter-band material can
easily be taken up).
10. The chromoplast pellet is very unstable at this particular stage.
To minimize chromoplast losses perform decantation of the
supernatant immediately after removing the tube from the
centrifuge by carefully pouring the supernatant in the direction
opposed to the pellet.
11. Chromoplast resuspension is more effective if initially done
into a smaller volume before completing to the full volume.
This forces any chromoplast clumps to pass more frequently
through the pipette tip opening, favoring their disaggregation.
Chromoplast Fractionation
195