Biology Reference
In-Depth Information
8. Dialysis cup (12,000 MWCO) (Cosmo Bio, Tokyo, Japan).
9. Receptacle tube (Maruemu yohki No. 2) (Maruemu, Osaka,
Japan).
10. [
14
C]Myristic acid (American Radiolabeled Chemicals, St
Louis, MO).
11. 3.75 mM myristic acid: Dissolve 8.56 mg of myristic acid
(Nacalai tesque) in 10 ml ethanol.
12. [
14
C]Leucine (PerkinElmer Japan, Yokohama, Japan).
2.4 Functional
Analysis of AtDTC
1. 2× Substrate solution: 60 mM PIPES-NaOH, pH 6.5, 15 mM
potassium gluconate, 50 mM 2-oxoglutarate.
2. Dowex AG 1 × 8 (100-200 mesh) (Bio-Rad, Tokyo, Japan).
3. Pasteur pipette (5 3/4
″
) (Asahi Glass Co., Ltd., Yokohama,
Japan).
4. Equilibration solution: 10 mM PIPES-NaOH, pH 6.5,
40 mM potassium gluconate, 100 mM sodium gluconate.
5. [
14
C]2-Oxoglutarate (PerkinElmer Japan).
6. 2-Oxoglutarate solution: 4.2 mM 2-oxoglutarate (3 Ci/mol).
7. Stop solution: 360 mM pyridoxal 5
′
-phosphate, 64 mM mer-
salyl acid.
2.5 Purifi cation
of bR
1. Ni-NTA-agarose (Qiagen, Tokyo, Japan).
2.
n
-Dodecyl-
-
D
-maltopyranoside (Anatrace).
3. Buffer A: 50 mM sodium phosphate, pH 7.4, 300 mM NaCl,
20 mM imidazole, and 0.15 %
n
-dodecyl-
β
-
D
-maltopyranoside.
4. Buffer B: 50 mM sodium phosphate, pH 7.4, 300 mM NaCl,
50 mM imidazole, and 0.15 %
n
-dodecyl-
β
-
D
-maltopyranoside.
5. Buffer C: 50 mM sodium phosphate, pH 7.4, 300 mM NaCl,
250 mM imidazole, and 0.05 %
n
-dodecyl-
β
-
D
-maltopyranoside.
6. Poly-Prep Chromatography Column (Bio-Rad, Richmond, CA).
β
2.6 Radio Isotope
Imaging
1. Imaging analyzer (BAS2000 imaging system) (Fuji Photo Film
Co., Ltd., Tokyo, Japan).
2. BAS cassette2 2040 (Fuji Photo Film Co., Ltd).
3. Imaging plate (BAS-MS 2040) (Fuji Photo Film Co., Ltd).
1. Accudenz (Accurate Chemical and Scientifi c, Westbury, NY).
2. Ultracentrifugation buffer: 30 mM HEPES-KOH, pH 7.8,
100 mM potassium acetate, 2.7 mM magnesium acetate. Store
at room temperature.
3. Ultracentrifugation tube (2.2 PA) and rotor (S55S) (Hitachi
Koki, Tokyo, Japan).
2.7 Accudenz
Density Gradient
Ultracentrifugation
