6. Chemically Defined Medium (CDM): 10 g/L glucose, 4.5 g/
L sodium acetate 3H 2 O, 3.195 g/L sodium phosphate monobasic H 2 O, 7.350 sodium phosphate dibasic anhydrous, 1 g/L
potassium phosphate monobasic, 0.2 g/L potassium phosphate dibasic, 0.7 g/L magnesium sulfate heptahydrate,
0.005 g/L manganese sulfate anhydrous, 0.005 g/L ferrous
sulfate heptahydrate, 0.001 g/L ferric nitrate 9H 2 O,
0.0051 g/L calcium chloride anhydrous, 0.1 g/L DL-alanine,
0.1 g/L L-arginine free base, 0.1 g/L L-aspartic acid, 0.5 g/L
L-cysteine free base, 0.05 g/L L-cysteine, 2HCl, 0.1 g/L Lglutamic acid, 0.2 g/L L-glutamine, 0.1 g/L glycine, 0.1 g/L
L-histidine free base, 0.1 g/L trans-4-hydroxy L-proline,
0.1 g/L L-isoleucine, 0.1 g/L L-leucine, 0.1 g/L L-lysine,
0.1 g/L L-methionine, 0.1 g/L L-phenylalanine, 0.1 g/L Lproline, 0.1 g/L L-serine, 0.2 g/L L-threonine, 0.1 g/L Ltryptophan, 0.1 g/L L-tyrosine free base, 0.1 g/L L-valine,
0.02 g/L adenine free base, 0.02 g/L guanine hydrochloride,
0.02 g/L uracil, 0.0002 g/L PABA (4-aminobenzoic acid),
0.0002 g/L biotin, 0.0008 g/L folic acid, 0.001 g/L niacinamide, 0.0025 g/L B-NAD (β-nicotinamide adenine dinucleotide hydrate), 0.002 g/L D-Ca pantothenate (calcium-Dpantothenate), 0.001 g/L pyridoxal hydrochloride, 0.001 g/
L pyridoxamine dihydrochloride, 0.002 g/L riboflavin,
0.001 g/L thiamine HCl, 0.0001 g/L cyanocobalamin, 5 g/
L sodium bicarbonate, 1 g/L choline chloride, 0.75 g/L Lcysteine hydrochloride.
7. Isopropyl β-D-1-thiogalactopyranoside (IPTG): 100 mM
stock.
8. 40% glycerol–water solution.
2.3 Protein
Purification Materials
1. HisTrap HP column, GE Healthcare.
2. Buffer A: 50 mM Na 2 HPO 4 , 500 mM NaCl, and 10% glycerol.
3. Buffer B: 50 mM Na 2 HPO 4 , 500 mM NaCl, 10% glycerol, and
250 mM imidazole.
4. 100 mM NiSO 4 .
5. 50 mM ethylenediaminetetraacetic acid (EDTA).
6. 1 M guanidinium hydrochloride.
7. 20% ethanol.
8. Sodium dodecyl sulfate.
9. 30% acrylamide–bis solution, 29:1.
10. Ammonium persulfate (APS): 10% solution.
11. 10Â Tris–glycine–SDS (25 mM Tris, 192 mM glycine, and
0.1% SDS), pH 8.3.
12. Tris–HCl: 0.5 M, pH 6.8.
464
Ruiquan Qi et al.
L sodium acetate 3H 2 O, 3.195 g/L sodium phosphate monobasic H 2 O, 7.350 sodium phosphate dibasic anhydrous, 1 g/L
potassium phosphate monobasic, 0.2 g/L potassium phosphate dibasic, 0.7 g/L magnesium sulfate heptahydrate,
0.005 g/L manganese sulfate anhydrous, 0.005 g/L ferrous
sulfate heptahydrate, 0.001 g/L ferric nitrate 9H 2 O,
0.0051 g/L calcium chloride anhydrous, 0.1 g/L DL-alanine,
0.1 g/L L-arginine free base, 0.1 g/L L-aspartic acid, 0.5 g/L
L-cysteine free base, 0.05 g/L L-cysteine, 2HCl, 0.1 g/L Lglutamic acid, 0.2 g/L L-glutamine, 0.1 g/L glycine, 0.1 g/L
L-histidine free base, 0.1 g/L trans-4-hydroxy L-proline,
0.1 g/L L-isoleucine, 0.1 g/L L-leucine, 0.1 g/L L-lysine,
0.1 g/L L-methionine, 0.1 g/L L-phenylalanine, 0.1 g/L Lproline, 0.1 g/L L-serine, 0.2 g/L L-threonine, 0.1 g/L Ltryptophan, 0.1 g/L L-tyrosine free base, 0.1 g/L L-valine,
0.02 g/L adenine free base, 0.02 g/L guanine hydrochloride,
0.02 g/L uracil, 0.0002 g/L PABA (4-aminobenzoic acid),
0.0002 g/L biotin, 0.0008 g/L folic acid, 0.001 g/L niacinamide, 0.0025 g/L B-NAD (β-nicotinamide adenine dinucleotide hydrate), 0.002 g/L D-Ca pantothenate (calcium-Dpantothenate), 0.001 g/L pyridoxal hydrochloride, 0.001 g/
L pyridoxamine dihydrochloride, 0.002 g/L riboflavin,
0.001 g/L thiamine HCl, 0.0001 g/L cyanocobalamin, 5 g/
L sodium bicarbonate, 1 g/L choline chloride, 0.75 g/L Lcysteine hydrochloride.
7. Isopropyl β-D-1-thiogalactopyranoside (IPTG): 100 mM
stock.
8. 40% glycerol–water solution.
2.3 Protein
Purification Materials
1. HisTrap HP column, GE Healthcare.
2. Buffer A: 50 mM Na 2 HPO 4 , 500 mM NaCl, and 10% glycerol.
3. Buffer B: 50 mM Na 2 HPO 4 , 500 mM NaCl, 10% glycerol, and
250 mM imidazole.
4. 100 mM NiSO 4 .
5. 50 mM ethylenediaminetetraacetic acid (EDTA).
6. 1 M guanidinium hydrochloride.
7. 20% ethanol.
8. Sodium dodecyl sulfate.
9. 30% acrylamide–bis solution, 29:1.
10. Ammonium persulfate (APS): 10% solution.
11. 10Â Tris–glycine–SDS (25 mM Tris, 192 mM glycine, and
0.1% SDS), pH 8.3.
12. Tris–HCl: 0.5 M, pH 6.8.
464
Ruiquan Qi et al.