8. Transfer the silica powder containing the carotenoid from the
aluminum foil into the glass tube containing the 4 mL of
acetone.
9. Allow the solution to sit for 10 min to let the white silica
powder settle to the bottom of the glass tube, leaving a colored
carotenoid solution at the top.
10. Carefully transfer the carotenoid acetone solution into a
collection tube.
11. Using the concentrator, dry vacuum the content of the Eppendorf tubes for about 20 min or until liquid has evaporated.
12. Redissolve the carotenoid substrate in an appropriate solvent
and determine the optical density of the carotenoid in a spectrometer (see Note 14). Use the molar extinction coefficient to
calculate the molar concentration of the carotenoid.
3.6 Enzymatic Assay
for Recombinant CCDs
1. Set thermomixer to 35
C and 600 rpm.
2. Mix 33 μL of 3% (w/v) of DMN with the appropriate amount
of carotenoid substrate volume (see Note 14).
3. Using the concentrator, dry vacuum the samples for
10–20 min, until liquid has completely evaporated.
4. Pipet 100 μL of either the soluble protein extract or purified
enzyme solution into the Eppendorf tube containing the dried
carotenoid/detergent residue.
5. Vortex vigorously for 20–30 s to form micelles.
6. Place samples on the thermomixer and incubate for various
time points. The progress of the enzymatic reaction is indicated
by a color shift of the solution from yellow to pale (Fig. 3).
7. Stop the reaction by adding in the following order 100 μL
distilled water, 400 μL acetone, 400 μL DEE, and 100 μL PE.
8. Using a table top centrifuge, centrifuge for 30 s at 6800 Â g at
room temperature to separate the phases.
9. Transfer the solvent layer (top layer), which should be ~600 μL
to a new labeled tube.
10. Using a concentrator, dry vacuum samples for ~10–20 min or
until liquid has completely evaporated.
11. Redissolve the dried pellet in running agents and analyze product formation by high-performance liquid chromatography
(HPLC) analysis (Fig. 4) (see Note 15).
Mammalian Carotenoid Cleavage Dioxygenases
83
aluminum foil into the glass tube containing the 4 mL of
acetone.
9. Allow the solution to sit for 10 min to let the white silica
powder settle to the bottom of the glass tube, leaving a colored
carotenoid solution at the top.
10. Carefully transfer the carotenoid acetone solution into a
collection tube.
11. Using the concentrator, dry vacuum the content of the Eppendorf tubes for about 20 min or until liquid has evaporated.
12. Redissolve the carotenoid substrate in an appropriate solvent
and determine the optical density of the carotenoid in a spectrometer (see Note 14). Use the molar extinction coefficient to
calculate the molar concentration of the carotenoid.
3.6 Enzymatic Assay
for Recombinant CCDs
1. Set thermomixer to 35
C and 600 rpm.
2. Mix 33 μL of 3% (w/v) of DMN with the appropriate amount
of carotenoid substrate volume (see Note 14).
3. Using the concentrator, dry vacuum the samples for
10–20 min, until liquid has completely evaporated.
4. Pipet 100 μL of either the soluble protein extract or purified
enzyme solution into the Eppendorf tube containing the dried
carotenoid/detergent residue.
5. Vortex vigorously for 20–30 s to form micelles.
6. Place samples on the thermomixer and incubate for various
time points. The progress of the enzymatic reaction is indicated
by a color shift of the solution from yellow to pale (Fig. 3).
7. Stop the reaction by adding in the following order 100 μL
distilled water, 400 μL acetone, 400 μL DEE, and 100 μL PE.
8. Using a table top centrifuge, centrifuge for 30 s at 6800 Â g at
room temperature to separate the phases.
9. Transfer the solvent layer (top layer), which should be ~600 μL
to a new labeled tube.
10. Using a concentrator, dry vacuum samples for ~10–20 min or
until liquid has completely evaporated.
11. Redissolve the dried pellet in running agents and analyze product formation by high-performance liquid chromatography
(HPLC) analysis (Fig. 4) (see Note 15).
Mammalian Carotenoid Cleavage Dioxygenases
83