5. Vortex 10 s and put the vials in a homogenizer.
6. Shake with the homogenizer for 30 s at 15–20 Hz.
7. Keep at room temperature for 5 min.
8. Repeat steps 5–7.
9. Centrifuge at 20,000 Â g for 20 min.
10. Transfer 600 μL of supernatant to HPLC filter tubes (e.g.,
Mini-Uniprep PTFE filter).
11. Take 50 μL, add formic acid (0.1% v/v) and internal standard
and characterize apocarotenoid content by HPLC-PDAHRMS (see Notes 7 and 8).
12. Use a freshly prepared plant extract to perform transport
assays. Depending on the stability of the analytes, the extract
can be stored at 4
C for 1–2 days.
3.5 Transportomics
Assay Using the Rapid
Filtration Technique
[20]
1. Thaw microsomes on ice.
2. Calculate the total protein content of microsome preparations
by Bradford assay [21]. Generally, “good” microsome preparations contain !400 μg of total proteins/100 μL of microsomes
with an OD 600 ¼ 4). If necessary, dilute microsomes to appropriate concentration.
3. Prepare two reaction mixtures on ice, one minus and one plus
ATP. The minus ATP mixture contains 1 mM DTT, 1 mM
MgSO 4 , 100 μg/mL creatine kinase, 10 mM creatine phosphate, and 10–20 μL of total plant extract in transport buffer
+DTT (see Note 6). The plus ATP mixture contains all of the
above, plus 5 mM MgSO 4 and 4 mM MgATP. Aliquot 550 μL
per reaction and incubate 5–10 min at 24
C.
4. Start the reactions by adding 100 μL of microsomes (final
volume of reaction ¼ 650 μL). Incubate the assays at 24
C.
5. Stop the reaction at different time points (at least one after 30 s
and one after 15 min) by loading 100 μL of the reaction
mixture on a prewetted nitrocellulose filter (0.45 μm pore
size) placed under vacuum (around 25 inches Hg) on a vacuum
manifold. Perform three technical replicates for each time
point. As soon as the mixture has been filtered, immediately
wash the filter three times with 2.5 mL of ice-cold transport
buffer (see Note 9).
6. Recover the filters and place on a paper towel. Let the filters dry
out completely at room temperature.
7. Place the filters inside a 2 mL amber glass vial.
3.6 Analysis
of Transport by HPLCPDA-HRMS
1. For the analysis of polar and semipolar substrate dissolve the
filter-bound microsomes by adding 1 mL of cold 75% (v/v)
96
Olivia Costantina Demurtas et al.
6. Shake with the homogenizer for 30 s at 15–20 Hz.
7. Keep at room temperature for 5 min.
8. Repeat steps 5–7.
9. Centrifuge at 20,000 Â g for 20 min.
10. Transfer 600 μL of supernatant to HPLC filter tubes (e.g.,
Mini-Uniprep PTFE filter).
11. Take 50 μL, add formic acid (0.1% v/v) and internal standard
and characterize apocarotenoid content by HPLC-PDAHRMS (see Notes 7 and 8).
12. Use a freshly prepared plant extract to perform transport
assays. Depending on the stability of the analytes, the extract
can be stored at 4
C for 1–2 days.
3.5 Transportomics
Assay Using the Rapid
Filtration Technique
[20]
1. Thaw microsomes on ice.
2. Calculate the total protein content of microsome preparations
by Bradford assay [21]. Generally, “good” microsome preparations contain !400 μg of total proteins/100 μL of microsomes
with an OD 600 ¼ 4). If necessary, dilute microsomes to appropriate concentration.
3. Prepare two reaction mixtures on ice, one minus and one plus
ATP. The minus ATP mixture contains 1 mM DTT, 1 mM
MgSO 4 , 100 μg/mL creatine kinase, 10 mM creatine phosphate, and 10–20 μL of total plant extract in transport buffer
+DTT (see Note 6). The plus ATP mixture contains all of the
above, plus 5 mM MgSO 4 and 4 mM MgATP. Aliquot 550 μL
per reaction and incubate 5–10 min at 24
C.
4. Start the reactions by adding 100 μL of microsomes (final
volume of reaction ¼ 650 μL). Incubate the assays at 24
C.
5. Stop the reaction at different time points (at least one after 30 s
and one after 15 min) by loading 100 μL of the reaction
mixture on a prewetted nitrocellulose filter (0.45 μm pore
size) placed under vacuum (around 25 inches Hg) on a vacuum
manifold. Perform three technical replicates for each time
point. As soon as the mixture has been filtered, immediately
wash the filter three times with 2.5 mL of ice-cold transport
buffer (see Note 9).
6. Recover the filters and place on a paper towel. Let the filters dry
out completely at room temperature.
7. Place the filters inside a 2 mL amber glass vial.
3.6 Analysis
of Transport by HPLCPDA-HRMS
1. For the analysis of polar and semipolar substrate dissolve the
filter-bound microsomes by adding 1 mL of cold 75% (v/v)
96
Olivia Costantina Demurtas et al.