2. Set up an overnight culture of ER2738 E. coli cells. Pick a single
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm. If performing competitive panning, set this up at
the end of day 2.
Day 2
3. Wash the blocked wells 3Â with 300 μl of PBST and replace
with 100 μl per well of 2Â Blocking Buffer.
4. Immobilization of biotinylated target and deselection target
(if using) (see Note 6). Add 1 μg of biotinylated target to the
selection well (see Note 7). If required, add 1 μg of biotinylated
deselection target to the negative control well and pre-pan
wells 1, 2, 3, and 4. Cover with a plate seal and incubate for
10 min at room temperature on a vibrating platform shaker.
5. Pre-pan 1. Remove the blocking buffer/deselection target
from pre-pan well 1. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Add 200 μl of phage-containing
supernatant from panning round 2 and 20 μl of 10Â Blocking
Buffer. Cover with a plate seal and incubate for 1 h at room
temperature on the vibrating platform shaker.
6. Pre-pan 2. Remove the blocking buffer/deselection target
from pre-pan well 2. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 1 to pre-pan well 2. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker.
7. Pre-pan 3. Remove the blocking buffer/deselection target
from pre-pan well 3. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 2 to pre-pan well 3. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker.
8. Pre-pan 4. Remove the blocking buffer/deselection target
from pre-pan well 4. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 3 to pre-pan well 4. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker. If
required, add 1 μg of non-biotinylated deselection target to the
pre-panned phage prior to selection (see Note 3).
9. Selection. Wash the selection well containing the immobilized
target and the negative control well 6Â with 300 μl of PBST.
Transfer 100 μl of pre-panned phage from pre-pan well 4 into
the selection well and 100 μl into the negative control well.
Cover with a plate seal and incubate for 30 min at room
temperature on the vibrating platform shaker.
116
Anna A. S. Tang et al.
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm. If performing competitive panning, set this up at
the end of day 2.
Day 2
3. Wash the blocked wells 3Â with 300 μl of PBST and replace
with 100 μl per well of 2Â Blocking Buffer.
4. Immobilization of biotinylated target and deselection target
(if using) (see Note 6). Add 1 μg of biotinylated target to the
selection well (see Note 7). If required, add 1 μg of biotinylated
deselection target to the negative control well and pre-pan
wells 1, 2, 3, and 4. Cover with a plate seal and incubate for
10 min at room temperature on a vibrating platform shaker.
5. Pre-pan 1. Remove the blocking buffer/deselection target
from pre-pan well 1. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Add 200 μl of phage-containing
supernatant from panning round 2 and 20 μl of 10Â Blocking
Buffer. Cover with a plate seal and incubate for 1 h at room
temperature on the vibrating platform shaker.
6. Pre-pan 2. Remove the blocking buffer/deselection target
from pre-pan well 2. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 1 to pre-pan well 2. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker.
7. Pre-pan 3. Remove the blocking buffer/deselection target
from pre-pan well 3. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 2 to pre-pan well 3. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker.
8. Pre-pan 4. Remove the blocking buffer/deselection target
from pre-pan well 4. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 3 to pre-pan well 4. Cover with a plate seal and incubate
for 1 h at room temperature on the vibrating platform shaker. If
required, add 1 μg of non-biotinylated deselection target to the
pre-panned phage prior to selection (see Note 3).
9. Selection. Wash the selection well containing the immobilized
target and the negative control well 6Â with 300 μl of PBST.
Transfer 100 μl of pre-panned phage from pre-pan well 4 into
the selection well and 100 μl into the negative control well.
Cover with a plate seal and incubate for 30 min at room
temperature on the vibrating platform shaker.
116
Anna A. S. Tang et al.