Protein Protocols Protein Protocols

1074 Dörsam et al.
with the phagemid vector to which the PCR products have been ligated. The phagemidcontaining
bacteria are infected with a helper phage such as M13KO7 to yield recombinant
phage particles that display scFv or Fab fragments fused to the pIII protein.
Phage displaying antibody fragments that bind to a specific antigen can be enriched from a
large library of phages by panning over the antigen. Nonspecific phages are removed
during the washing procedure, following in which the remaining antigen-specific phages
are eluted and used to reinfect exponentially growing E. coli. The specificity of the
enriched phage particles can be confirmed in an enzyme-linked immunosorbent assay
(ELISA). The entire procedure, which is described in this chapter, is outlined on a flow
chart (Fig. 1).
2. Materials
2.1. Phage Rescue, Panning, Reinfection, Small-Scale Phage Rescue
1. Sterile glass 100-mL and 1000-mL Erlenmeyer flasks.
2. Petri dishes, 85-mm and 145-mm diameter (Greiner, Frickenhausen, Germany).
3. Maxisorb ® Immunotubes and caps (Nunc, Roskilde, Denmark).
4. Sterile polypropylene 96-deep-well microtiter plate (Beckmann, München, Germany) and
sealing device (Beckman, Biomek, cat. no. 538619).
5. End over end shaker.
6. Tabletop microcentrifuge.
7. Clinical centrifuge with swinging-bucket rotor.
8. IEC refrigerated centrifuge (or equivalent) with adaptors for microtiter plates.
9. ELISA plate shaker (e.g., IKA MTS-2).
10. Helper phage M13KO7-derived VCSM13 (Stratagene cloning systems, La Jolla, CA).
11. XL-1 Blue bacteria (Stratagene cloning systems, La Jolla, CA).
12. 2 M Glucose, sterile filtered.
13. (5 mg/mL) Ampicillin, sterile filtered.
14. (10 mg/mL) Kanamycin, sterile filtered.
15. M9-minimal medium agar plates: To 15 g of Bacto-agar add water to 750 mL and autoclave.
After the solution has cooled to 55°C, add 200 mL of sterile 5× M9 salts, 50 mL of
sterile 2 M glucose, and 10 mL of sterile-filtered 100× supplement. Pour plates quickly.
16. 5× M9 salts: To 37.4 g of Na2HPO4·2H2O, 11.75 g of NaH2PO4·H2O, 2.5 g of NaCl, and
5 g of NH4Cl, add distilled water to 1 L and autoclave.
17. 100× Supplement: To 1 mL of 1 M MgSO4·7H2O, 1 mL of 100 mM CaCl2·2 H2O, 30 µL
of 100 mM Fe(III)Cl3·6H2O and 60 µL of 500 mM thiamin, add distilled water to 10 mL
and sterile filter.
18. SOB-GA medium agar plates: To 15 g of Bacto-agar, 20 g of Bacto-tryptone, 5 g of Bactoyeast
extract, and 0.5 g of NaCl, add distilled water to 920 mL and autoclave. After the
medium has cooled to 55°C, add 10 mL of sterile MgCl2, 50 mL of sterile 2 M glucose,
and 20 mL of sterile filtered ampicillin (5 mg/mL). Pour plates quickly.
19. 2× YT Medium: Dissolve 15 g of Bacto-agar, 17 g of Bacto-tryptone, 10 g of Bacto-yeast
extract and 5 g of NaCl in 800 mL of distilled water, adjust the pH of the medium to
7.5 with 1 M NaOH, add distilled water to 1 L total volume and autoclave.
20. 2× YT-GA Medium: 2× YT medium containing 100 µg/mL of Ampicillin and 100 mM
glucose.
21. 2× YT-AK Medium: 2× YT medium containing 100 µg/mL of Ampicillin and 50 µg/mL
of Kanamycin.