6. Repeat step 4.
7. Dissolve the pellet in sterile bd water (600 mL).
8. Repeat steps 4 and 7.
9. Repeat step 4 and dissolve the pellet in digestion medium; the
volume of digestion medium is established as such that OD 600
is 10.
10. Incubate the cells in digestion medium at 30
C, continuous
shaking.
11. After 40 min establish the ratio OD 600 in digestion medium/
OD 600 in water. If the ratio is approximately 3, stop the digestion; if not, continue the digestion reaction and measure again
the ratio OD 600 in digestion medium:OD 600 in water after
30 min; stop digestion after 2 h at the latest.
12. Centrifuge the digestion mix for 10 min at 1200 Â g and start
working on ice.
13. Resuspend the pellet in 25 mL homogenization buffer.
14. Disrupt cells via homogenization (35–50 Â up/down in a glass
potter homogenizer).
15. Pellet the disrupted cells by centrifugation at 6000 Â g,
10 min, 4
C.
16. Collect supernatant in ultracentrifuge tubes and resuspend
pellet in 10 mL homogenization buffer.
17. Repeat disruption (35–50 Â up/down in the glass potter).
18. Repeat step 15.
19. Recover the supernatant and transfer to the ultracentrifuge
tubes containing the supernatant of the previous
centrifugation.
20. Pellet down total microsomes in an ultracentrifuge
(100,000 Â g, 45 min, 4
C).
21. Resuspend pellet in storage buffer to a final OD 600 of 4.
22. Aliquot microsomes into reaction tubes (250 μL/tube).
23. Snap-freeze tubes in liquid nitrogen, and store them at
À80
C.
3.4 Total Plant
Extract
for Transportomics
Assay (Quantity
for 25–50 Assays)
1. Lyophilize plant material in a freeze-dryer.
2. Reduce the dried material in powder by agitation in the presence of tungsten beads in a homogenizer or by mortar and
pestle.
3. Weight 3–30 mg powder according to the plant matrix.
4. For extraction of polar and semipolar metabolites add 750 μL
of cold 50–75% (v/v) LC-MS grade methanol (see Notes 5
and 8).
Transportomics for Apocarotenoid Transporters
95
7. Dissolve the pellet in sterile bd water (600 mL).
8. Repeat steps 4 and 7.
9. Repeat step 4 and dissolve the pellet in digestion medium; the
volume of digestion medium is established as such that OD 600
is 10.
10. Incubate the cells in digestion medium at 30
C, continuous
shaking.
11. After 40 min establish the ratio OD 600 in digestion medium/
OD 600 in water. If the ratio is approximately 3, stop the digestion; if not, continue the digestion reaction and measure again
the ratio OD 600 in digestion medium:OD 600 in water after
30 min; stop digestion after 2 h at the latest.
12. Centrifuge the digestion mix for 10 min at 1200 Â g and start
working on ice.
13. Resuspend the pellet in 25 mL homogenization buffer.
14. Disrupt cells via homogenization (35–50 Â up/down in a glass
potter homogenizer).
15. Pellet the disrupted cells by centrifugation at 6000 Â g,
10 min, 4
C.
16. Collect supernatant in ultracentrifuge tubes and resuspend
pellet in 10 mL homogenization buffer.
17. Repeat disruption (35–50 Â up/down in the glass potter).
18. Repeat step 15.
19. Recover the supernatant and transfer to the ultracentrifuge
tubes containing the supernatant of the previous
centrifugation.
20. Pellet down total microsomes in an ultracentrifuge
(100,000 Â g, 45 min, 4
C).
21. Resuspend pellet in storage buffer to a final OD 600 of 4.
22. Aliquot microsomes into reaction tubes (250 μL/tube).
23. Snap-freeze tubes in liquid nitrogen, and store them at
À80
C.
3.4 Total Plant
Extract
for Transportomics
Assay (Quantity
for 25–50 Assays)
1. Lyophilize plant material in a freeze-dryer.
2. Reduce the dried material in powder by agitation in the presence of tungsten beads in a homogenizer or by mortar and
pestle.
3. Weight 3–30 mg powder according to the plant matrix.
4. For extraction of polar and semipolar metabolites add 750 μL
of cold 50–75% (v/v) LC-MS grade methanol (see Notes 5
and 8).
Transportomics for Apocarotenoid Transporters
95