– 100Â His: in 100 mL bd H 2 O, dissolve 0.6 g of L-histidine
(1.2 g/200 mL).
– 100Â Leu: in 100 mL bd H 2 O, dissolve 2.6 g of L-leucine
(0.8 g/200 mL).
These three stocks can be autoclaved at 121
C for 20 min
and then store at 4
C.
– 100Â Trp: in 100 mL bd H 2 O dissolve 0.8 g of L-tryptophan
(4 g/500 mL). Do not autoclave. Filter-sterilize (0.22 μm),
store at 4
C in the dark.
2.3 Solutions
for Microsome
Preparation
– Digestion medium: 1.1 M sorbitol, 20 mM Tris. Adjust pH to
7.6 with 6 M HCl. Just before starting the digestion step add
1 mM DTT and 57 U Lyticase/mL digestion buffer (Lyticase
from Arthrobacter luteus) (see Note 1).
– Homogenization buffer: 1.1 M Glycerol anhydrous, 50 mM
Tris, 5 mM EDTA. Adjust pH to 7.4 with ascorbic acid (powder). Just before starting the homogenization step freshly add
1 mM DTT, 1.5% (w/v) PVP [Polyvinylpyrrolidone (K30),
1000 (water max 5%)], 2 mg/mL BSA (Albumin, from bovine
serum), 1 mM PMSF (100 or 200 mM stock solution in 70%
ethanol) (see Note 1).
– Storage buffer: 1.1 M glycerol anhydrous, 50 mM Tris, 1 M
EDTA. Adjust pH to 7.4 with MES (powder). Just before starting the storage step freshly add: 1 mM DTT, 2 mg/mL BSA,
1 mM PMSF (see Note 1).
2.4 Solutions for In
Vitro Transport Assays
– Transport buffer: 0.4 M Glycerol anhydrous, 100 mM KCl,
20 mM Tris. Adjust pH to 7.4 with MES (powder) (see Note 1).
– Transport buffer + DTT: transport buffer freshly supplemented
with 1 mM DTT (see Note 1).
2.5 Equipment
1. Incubator.
2. Spectrophotometer.
3. Electroporator.
4. Ultracentrifuge.
5. Glass Potter homogenizer.
6. Vacuum centrifuge.
7. Liquid nitrogen.
8. Vacuum manifold.
9. Shaker with controlled temperature.
10. Biological hood.
11. Vortex.
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