4. Add approx. 1 mL of the soluble fraction of the bacterial lysate
to the micro spin column (see Note 13).
5. Mix by pipetting up and down five times. Incubate for up to
5 min.
6. Centrifuge at 1000 Â g for 1 min to remove the unbound
proteins.
7. Add at least 5 column volumes of wash buffer and pipet up and
down five times. Centrifuge at 1000 Â g for 1 min to remove
the remaining unbound proteins (the wash step can be
repeated if necessary).
8. Add at least 5 column volumes of elution buffer and pipet up
and down five times. Incubate for 5 min and harvest the protein
by centrifugation at 1000 Â g for 1 min.
9. Transfer the purified protein to an Amicon
® Ultra—0.5 mL
10 kD centrifugal filter. Concentrate the protein solution.
Dilute the concentrated protein solution with extraction
buffer containing 1mM final concentration of TCEP if
necessary.
10. The purity of the recombinant protein can be analyzed by
SDS-PAGE and Coomassie brilliant blue staining using standard protocols.
3.5 Carotenoid
Substrate Preparation
for Enzymatic Assay
1. In the TLC developing tank, place the following: 40 mL of PE,
10 mL of acetone, and 10 mL of DEE. Mix the contents that
are in the TLC developing tank and cover with glass cover to
prevent solvent vapors from escaping.
2. Take one TLC silica plate and use a ruler and a pencil to draw a
straight line that is 2.54 cm (1 inch) from the bottom of the
TLC silica plate.
3. Place about 1 mg of carotenoid powder into an Eppendorf
tube and immediately add 200–300 μL of chloroform and
mix to dissolve.
4. Using a pipette tip, distribute the dissolved carotenoid chloroform solution on the pencil line and let the chloroform
evaporate.
5. Place the TLC silica plate into the TLC developing tank for
separation of the carotenoid. Separation of pure carotenoid
substrate from impurities is complete when the solvent reaches
the upper top of the TLC silica gel.
6. Add 4 mL of acetone into a glass tube.
7. Remove TLC silica plate from the TLC developing tank and
use a flat edge spatula to scrape the silica with carotenoid (large
yellow or red line) onto the aluminum foil.
82
Linda Dora Thomas et al.
to the micro spin column (see Note 13).
5. Mix by pipetting up and down five times. Incubate for up to
5 min.
6. Centrifuge at 1000 Â g for 1 min to remove the unbound
proteins.
7. Add at least 5 column volumes of wash buffer and pipet up and
down five times. Centrifuge at 1000 Â g for 1 min to remove
the remaining unbound proteins (the wash step can be
repeated if necessary).
8. Add at least 5 column volumes of elution buffer and pipet up
and down five times. Incubate for 5 min and harvest the protein
by centrifugation at 1000 Â g for 1 min.
9. Transfer the purified protein to an Amicon
® Ultra—0.5 mL
10 kD centrifugal filter. Concentrate the protein solution.
Dilute the concentrated protein solution with extraction
buffer containing 1mM final concentration of TCEP if
necessary.
10. The purity of the recombinant protein can be analyzed by
SDS-PAGE and Coomassie brilliant blue staining using standard protocols.
3.5 Carotenoid
Substrate Preparation
for Enzymatic Assay
1. In the TLC developing tank, place the following: 40 mL of PE,
10 mL of acetone, and 10 mL of DEE. Mix the contents that
are in the TLC developing tank and cover with glass cover to
prevent solvent vapors from escaping.
2. Take one TLC silica plate and use a ruler and a pencil to draw a
straight line that is 2.54 cm (1 inch) from the bottom of the
TLC silica plate.
3. Place about 1 mg of carotenoid powder into an Eppendorf
tube and immediately add 200–300 μL of chloroform and
mix to dissolve.
4. Using a pipette tip, distribute the dissolved carotenoid chloroform solution on the pencil line and let the chloroform
evaporate.
5. Place the TLC silica plate into the TLC developing tank for
separation of the carotenoid. Separation of pure carotenoid
substrate from impurities is complete when the solvent reaches
the upper top of the TLC silica gel.
6. Add 4 mL of acetone into a glass tube.
7. Remove TLC silica plate from the TLC developing tank and
use a flat edge spatula to scrape the silica with carotenoid (large
yellow or red line) onto the aluminum foil.
82
Linda Dora Thomas et al.