View - ResearchGate

116 Casey et al.
3. Wash the coated plate twice with PBS and block the plate with 200 l of 5%
blotto for 2–3 hr at room temperature.
4. Take an aliquot of the phage library and dilute to 10 11 phage/well in 1% blotto.
Allow the phage to incubate for 15 min in 1% blotto before adding to the plate
to remove the milk binding phage.
5. Wash the plate twice with PBS, then add 100 μl of the pre-incubated phage to
the blocked wells and incubate for 2–3 hr on the bench at room temperature.
6. When the K91 have grown to log phase remove from the shaking incubator and
allow to settle. This enables the F-pilus to regenerate.
7. Wash the ELISA plate using increased stringency per round of panning. For
example, use the following:
a. Round 1: 2 × PBS washes.
b. Round 2: 4 × PBST, then 2 × PBS.
c. Round 3: 6 × PBST, then 2 × PBS.
d. Round 4: 8 × PBST, then 2 × PBS.
8. Elute the bound phage by adding 100 μl elution buffer for 10 min, pool the
elutions and neutralize with equilibration buffer. Immediately add the pooled
phage to the stationary K91 culture and incubate for 1hat37ºC (mix gently
occasionally) to allow re-infection of the eluted phage.
9. Add the re-infected K91 culture to 200 ml SB media (containing 40 μg/ml
tetracycline) and expand the culture overnight at 37ºC (vigorous shaking).
10. Centrifuge the culture for 15 min at 4ºC at 10,400 g. Prepare glycerol stocks of
the pellet (final glycerol concentration 20%). Retain the supernatant and transfer
to a centrifuge tube and PEG precipitate overnight, using a 1:5 dilution of PEG
solution, shake and incubate on ice overnight in the cold room.
11. Spin the precipitated phage at 16,400 g for 50 min, resuspend in 1.5 ml PBS and
re-centrifuge at 15,700 g to remove remaining cell debris. Store phage at –80ºC.
12. Repeat steps 1–11 for subsequent rounds of panning.
3.2. Analyzing Rounds of Panning by ELISA
To ensure the panning process has been successful, an ELISA should be
performed. An example of the typical results obtained is shown in Fig. 2A.
1) Coat a microtiter plate (Nunc Maxisorp) with the antibody that was used for
panning using the same conditions (see Subheading 3.1.).
2) Wash and block as per panning conditions (see Subheading 3.1.).
3) Prepare phage dilutions of rounds 0–4, usually 10 10 /ml (see Subheading 3.3.) for
titration in PBS, apply 100 μl in duplicate wells and incubate for 1 h on a plate
shaker at room temperature. To check for non-specific binding, test for phage
binding to the blocking solution only or coat with an isotype control antibody.
4) Wash four times with PBST.