18. PSFM-J1/FBS-PS medium: PSFM-J1 medium, 2% FBS,
50,000 U/L penicillin G, 50 mg/L streptomycin.
19. 1Â Grace’s medium (Thermo Fisher Scientific).
20. FuGENE HD transfection reagent (Promega).
21. LUNA automated cell counter and cell counting slides
(Thermo Fisher Scientific).
22. Trypan blue: 0.4% solution in PBS, pH 7.2.
23. Six-well tissue plate with flat bottom.
24. 125-mL Erlenmeyer flask (Vent filter cap/Baffle bottom)
(Corning).
25. 3-L Fernbach flask (Vent filter cap/Baffle bottom) (Corning).
26. Orbital shaker incubator.
27. Centrifuge with adaptors for 1-L and 50-mL tubes.
28. Temperature-controlled room or incubator set at 27
C.
2.4 Protein
Purification
and Characterization
1. Ammonium sulfate.
2. TBS: 10 mM Tris-HCl (pH 7.5), 150 mM NaCl.
3. Buffer A: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM
imidazole.
4. Buffer B: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 250 mM
imidazole.
5. Buffer C: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM
imidazole.
6. Buffer D: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 500 mM
imidazole.
7. Ni-NTA Superflow resin (Qiagen).
8. Ni Sepharose excel resin (GE Healthcare).
9. Glass Econo-column (Bio-Rad).
10. HisTrap HP column (5 mL) (GE Healthcare).
11. HiLoad16/60 Superdex200 column (GE Healthcare).
12. Dialysis tubing (MWCO 10 K) (Spectra/Por).
13. Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator
(Merck).
14. Amicon Ultra-15 (MWCO 30 K) centrifugal concentrator
(Merck).
15. His 6 -tagged TEV protease (TEV-His 6 ) (see Note 2).
16. Chromatograhy system (e.g., BioLogic DuoFlow system
(Bio-Rad)).
Production of Recombinant Antibody Fragments via the iRAT system
83