Protein Protocols Protein Protocols

1080 Dörsam et al.
15. A negative control is always necessary to verify that an enrichment is due to binding of the
recombinant phage antibodies to the antigen rather than to components of the blocking buffer.
The tube used as negative control should be treated exactly like the antigen coated tubes.
16. The tube can be closed with Parafilm ® or with the caps provided by the supplier of
the tubes.
17. The amount of rescued phagemid antibodies used for panning is dependent on the complexity
of the library. Make sure that the number of phages is at least 10 3 –1 × 10 4 times
more than the complexity of the library.
18. To reduce nonspecific, hydrophobic protein–protein interactions between native M13 phage
proteins and some antigens, Triton X-100 may be added to a final concentration of 0.1%.
19. Incubation with triethylamine reduces the infectivity of the recombinant phage antibodies.
Do not exceed an incubation time of more than 5–10 min.
20. Handle the cells gently before and during infection with eluted phages.
21. This titration is to determine the number of infectious phagemid particles eluted from the
tube after panning. The number should be low after first round of panning and then
increase after subsequent rounds as specific clones are enriched.
22. It is very important that the agar plates are well dried before plating the bacteria.
23. If there are only a few colonies on the plates, extend the incubation at 30°C until the
colonies are fairly large. Resuspend the cells in a smaller volume.
24. After the third round of panning and reinfection, its possible to start analyzing single clones.
25. For the small-scale rescue of phagemids, we recommend the use of special polypropylene
96-deep-well microtiter plates (volume per well = 1 mL). However, these plates may
require a special adaptor for centrifugation.
26. If another shaker is used, speed may have to be decreased to prevent spillage of the
medium into adjacent wells.
27. The remaining master plate may be stored at 4°C for up to 2 wk. To store it longer, add
sterile glycerol to the cultures to a final concentration of 20%, seal the plate and mix by
inverting several times. Store at –70°C.
28. All incubation steps should be carried out in a wet chamber to prevent evaporation or
contamination. For example, the microwell plate can be placed in a closed box containing
moistened tissue paper.
29. These conditions work for most protein antigens or haptens bound to a protein carrier. The
optimal amount of antigen may vary. Nonprotein antigens (e.g., lipopolysaccharides) may
require a different buffer. In this case, refer to the buffers used in a conventional ELISA.
30. 2% Skimmed milk in PBS works very well as a blocking buffer. Some commercially
available blocking buffers (e.g., Pierce Superblock) give comparable results and should
be used according to the manufacturers instructions. If the solid support is coated with a
nonprotein antigen, other blocking buffers may be required. In this case, refer to the buffers
used in a conventional ELISA. Take note that collagen, FCS or gelatin do not work
well as blocking agents owing to their interaction with phagemid particles.
31. Both supernatant or PEG-precipitated phagemids may be used in phage ELISA. PEG precipitation
lowers the background and removes contaminants interferring with the binding.
32. The anti-M13 antibody is a polyclonal serum raised against M13 or fd phages. A commercial
source is listed in Subheading 2. The serum should be diluted according to the
manufacturer’s instructions in the same solution used for blocking as described in step 2.
The serum used here contains a high titer of antibodies against E. coli proteins and therefore
cross-reacts with proteins in the phage supernatant. To avoid this, the serum must be
preabsorbed. Incubate the diluted serum overnight with a piece of E. coli-lysate-coated
nitocellulose at 4°C. This can be done in parallel with the coating (Subheading 3.5., step 1).