Protein Protocols Protein Protocols

1066 Ehrlich, Berthold, and Bailon
1. Resuspend control insect cells (10 6 ) in 0.5 mL of Grace’s insect cell medium–2% nonfat
dry milk containing 1–2 × 10 11 bacteriophage library.
2. Incubate cells with bacteriophage library for 30 min at room temperature.
3. Sediment cells in a centrifuge at 1000g for 10 min.
4. Carefully remove supernatant containing bacteriophage.
5. Incubate supernatant with receptor bearing insect cells (10 6 ) for 30 min with gentle agitation.
6. Sediment cells in a centrifuge at 1000g for 5 min.
7. Discard supernatant.
8. Wash cells with 10 mL of Grace’s insect cell medium–2% nonfat dry milk.
9. Sediment cells in a centrifuge at 1000g for 5 min.
10. Discard supernatant.
11. Repeat steps 8–10 four more times.
12. Elute interacting phage with 0.5 mL of 6 M urea, pH 3, for 15 min at room temperature.
13. Neutralize eluate by adding 10 µL of 2 M Tris base.
14. Titer 1 µL (see Subheading 3.3.1.).
15. Amplify rest of eluate or store at 4°C (see Subheading 3.3.2.).
16. Resuspend receptor transfected mammalian cells (10 6 ) in 0.5 mL of HEPES buffer.
17. Add amplified eluate (1–2 × 10 11 pfu).
18. Incubate at room temperature for 30 min.
19. Sediment cells in a centrifuge at 1000g for 5 min.
20. Discard supernatant.
21. Wash cells with 10 mL of HEPES buffer.
22. Sediment cells in a centrifuge at 1000g for 5 min.
23. Discard supernatant.
24. Repeat steps 21–23 four more times.
25. Elute interacting phage with 0.5 mL of 6 M urea, pH 3.0, for 15 min at room temperature.
26. Neutralize eluate by adding 10 µL of 2 M Tris base.
27. Titer 1 µL (see Subheading 3.3.1.).
28. Amplify the rest of the eluate or store at 4°C (see Subheading 3.3.2.).
29. Repeat steps 5–14 using an input of 1 × 10 11 pfu.
30. Pick plaques for amplification and isolation of single stranded bacteriophage M13 DNA
(see Subheading 3.3.3.).
3.3. General Molecular Techniques
3.3.1. Phage Titering
Phage titering is a way of calculating the input and output of phage particles in
biopanning. This measurement is determined experimentally by infecting E. coli in a
petri dish and then evaluated by quantifying the number of plaque-forming units (pfu)
left overnight on the bacterial lawn.
1. Streak out XL1-Blue cells onto LB plates containing Tetracycline.
2. Invert plates.
3. Incubate at 37°C for 24 h.
4. Store (approx 1 mo) wrapped in parafilm at 4°C until needed.
5. Set up overnight culture by inoculating a single colony of XL1-Blue in LB broth (20 mL)
containing Tetracycline in a triple baffle shake flask (see Note 6).
6. Incubate at 37°C with vigorous shaking.
7. Dilute overnight culture 1100 in 20 mL of LB broth containing Tetracycline in another
shake flask.