Preparation of Isotopically Labeled Recombinant Fragments of Fibronectin for Functional and Structural Study by Heteronuclear Nuclear Magnetic Resonance Spectroscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 60

Fig. 2. Schematic representation of the fermentation vessel and controller. (1) tem-
perature-controlled jacket; (2) temperature probe; (3) nutrient-feed port; (4) base-con-
trol port; (5) acid-control port; (6) pH probe; (7) dO 2 probe; (8) impeller.

5. D-glucose solution: Prepare a 20 % (w/v) solution in water. Filter sterilize and

store at room temperature for up to 1 yr. Use U-[ 13 C]-glucose as necessary ( see
Note 3 ).

6. Antifoam solution: Autoclave polypropylene glycol 1025 and store at room tem-

perature indefinitely ( see Note 4 ).

7. Acid control solution: Prepare and autoclave 2 M orthophosphoric acid (corro-

sive) and store at room temperature indefinitely.

8. Base control solution: Prepare and autoclave 2 M potassium hydroxide (corro-

sive) and store at room temperature indefinitely.

2.3. Fermentation of P. pastoris
Methanol feed solution: Prepare fresh and filter sterilize 10% (v/v) metha-
nol (toxic) in water.

2.4. Processing of Culture Supernatants
1.

Citrate equilibration buffer pH 3.0 (per liter): Dissolve 3.15 g citric acid, 0.90 g
trisodium citrate dihydrate, and 0.20 g sodium azide (highly toxic) in water.

2. Citrate wash buffer pH 5.0 (per liter): Dissolve 1.34 g citric acid, 3.82 g triso-

dium citrate dihydrate, and 0.20 g sodium azide (highly toxic) in water.

3. Citrate elution buffer pH 5.0 (per liter): Dissolve 1.34 g citric acid, 3.82 g triso-

dium citrate dihydrate, 58.44 g sodium chloride, and 0.20 g sodium azide (highly
toxic) in water.