3.7.3 Selection Day 2–4
Repeat the selection steps using the out-phage form the previous
day as in-phage for the next round of selection. This procedure is
repeated for a total of four rounds.
1. Start 4 Â 10 ml E. coli OmniMAX cultures from the ON
culture stored in 4
C (see day 1, step 1).
2. Block each well of the preselection plate and the target coated
plate with 200 μl blocking solution for 1 h at 4
C with shaking.
3. Harvest phages from previous day: Pellet the bacteria by centrifugation (15 min, 3000 Â g).
4. Transfer the phage supernatants to fresh Falcon tubes supplemented with 1/5 final volume PEG/NaCl. Incubate for
15 min on ice. Centrifuge for 15 min at 16,000 Â g. Decant
the supernatant, then spin another 3 min at 16,000 Â g. Aspirate the remaining liquid. Resuspend the phage pellet in 1/10
volume (1 ml) cold PBS.
5. Continue with steps 4–13 of the protocol from day 1, using
100 μl resuspended phage pool as in-phages.
6. Analyze the progress of the selection by calculating the ratio of
out-phages versus in-phages for each day of selection.
7. Day 4: Immobilize protein (5–10 μg bait protein or
GST/MBP in 100 μl buffer) for analysis of the progress of
the selection through pooled phage ELISA (Fig. 2). Cover
the plates with a clear adhesive film and incubate overnight at
4
C.
3.7.4 Day 5:
Phage pool ELISA
After the last selection day, harvest the out-phages as described
above. Save 50 μl of the out-phage pools from each day of selection
in À20
C. Perform phage pool ELISA.
1. Block each well with blocking solution for 1 h at 4
C.
2. Wash the blocked plate four times with PT buffer.
3. Add 100 μl phage supernatant from the binding selections to
the corresponding well of the target-coated plate and the
GST/MBP-coated wells. Incubate the plates for 2 h at 4
C.
4. Wash five times with PT buffer.
5. To each well, add 100 μl anti-M13 antibody-HRP conjugate
(1:5000 dilution) in PBT buffer and incubate for 1 h at 4
C.
6. Wash four times with PT buffer and one time with PBS.
Remove as much liquid as possible by tapping the plate on
paper.
7. To each well, add 100 μl TMB substrate (consists of 1:1
TMB + peroxide solution mixed immediately before use).
Allow color to develop for 1-15 min (depending on how the
color develop) and stop the reaction by adding 100 μl 0.6 M
H 2 SO 4 .
Proteomic Peptide-Phage Display of PDZ Domains
55
Repeat the selection steps using the out-phage form the previous
day as in-phage for the next round of selection. This procedure is
repeated for a total of four rounds.
1. Start 4 Â 10 ml E. coli OmniMAX cultures from the ON
culture stored in 4
C (see day 1, step 1).
2. Block each well of the preselection plate and the target coated
plate with 200 μl blocking solution for 1 h at 4
C with shaking.
3. Harvest phages from previous day: Pellet the bacteria by centrifugation (15 min, 3000 Â g).
4. Transfer the phage supernatants to fresh Falcon tubes supplemented with 1/5 final volume PEG/NaCl. Incubate for
15 min on ice. Centrifuge for 15 min at 16,000 Â g. Decant
the supernatant, then spin another 3 min at 16,000 Â g. Aspirate the remaining liquid. Resuspend the phage pellet in 1/10
volume (1 ml) cold PBS.
5. Continue with steps 4–13 of the protocol from day 1, using
100 μl resuspended phage pool as in-phages.
6. Analyze the progress of the selection by calculating the ratio of
out-phages versus in-phages for each day of selection.
7. Day 4: Immobilize protein (5–10 μg bait protein or
GST/MBP in 100 μl buffer) for analysis of the progress of
the selection through pooled phage ELISA (Fig. 2). Cover
the plates with a clear adhesive film and incubate overnight at
4
C.
3.7.4 Day 5:
Phage pool ELISA
After the last selection day, harvest the out-phages as described
above. Save 50 μl of the out-phage pools from each day of selection
in À20
C. Perform phage pool ELISA.
1. Block each well with blocking solution for 1 h at 4
C.
2. Wash the blocked plate four times with PT buffer.
3. Add 100 μl phage supernatant from the binding selections to
the corresponding well of the target-coated plate and the
GST/MBP-coated wells. Incubate the plates for 2 h at 4
C.
4. Wash five times with PT buffer.
5. To each well, add 100 μl anti-M13 antibody-HRP conjugate
(1:5000 dilution) in PBT buffer and incubate for 1 h at 4
C.
6. Wash four times with PT buffer and one time with PBS.
Remove as much liquid as possible by tapping the plate on
paper.
7. To each well, add 100 μl TMB substrate (consists of 1:1
TMB + peroxide solution mixed immediately before use).
Allow color to develop for 1-15 min (depending on how the
color develop) and stop the reaction by adding 100 μl 0.6 M
H 2 SO 4 .
Proteomic Peptide-Phage Display of PDZ Domains
55