10. Wash the selection well and negative control well 27Â with
300 μl of PBST, preferably using an automated plate washer.
For standard panning, elute the phage immediately as
described in steps 13 and 14 (see Note 4). For competitive
panning, continue with the following step (see Note 5).
11. Add the following to the selection well and negative control
well: 80 μl of 2Â Blocking Buffer, 20 μl of 80% glycerol and 1 μl
of Halt protease inhibitor cocktail (100Â). If desired, add 1 μg
of non-biotinylated deselection target (see Note 3). Cover with
a plate seal and incubate for 22–24 h at room temperature on
the vibrating platform shaker.
Day 2 or 3
12. Wash the selection well and negative control well 6Â with
300 μl of PBST.
13. Elution 1. Add 100 μl of 0.2 M glycine, pH 2.2 to the selection
well and negative control well. Incubate for 10 min at room
temperature. Neutralize by adding 15 μl of 1 M Tris-HCl,
pH 9.1 and transfer the eluted phage to a
microcentrifuge tube.
14. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Add 100 μl of the diluted triethylamine to the selection well and
negative control well. Incubate for 6 min at room temperature.
Neutralize by adding 50 μl of 1 M Tris-HCl, pH 7 and transfer
the eluted phage to the microcentrifuge tube containing
elution 1.
15. Infection of ER2738 cells. Before eluting the phage, set up
5 ml cultures of ER2738 cells for each selection. Dilute the
overnight cultures of ER2738 cells in 2YT medium with
12 μg/ml tetracycline to an OD600 of ~0.2 and incubate at
37
C, 230 rpm for about 1 h until OD600 reaches ~0.6. Add
the eluted phage and incubate for 1 h at 37
C at up to 90 rpm.
Mix at least once during the incubation.
16. Plating the cells. Plate a range of volumes (e.g., 0.01 μl, 0.1 μl,
1 μl, 10 μl, and 100 μl) onto LB agar plates with 100 μg/ml
carbenicillin. Also, centrifuge and plate the remaining cells as
described for Panning Round 1 (see Subheading 3.1.1, step
13). For the negative control, select one volume to plate
(usually 10 μl). Incubate the plates overnight at 37
C.
Day 3 or 4
17. Compare the number of colonies on the negative control plate
with the selection plate containing the same volume of cells. A
successful screen shows enrichment of target-specific phage
with fewer colonies (less than half) on the negative control
plate. If background is still high on the negative control plate,
Isolation of Artificial Binding Proteins
117
300 μl of PBST, preferably using an automated plate washer.
For standard panning, elute the phage immediately as
described in steps 13 and 14 (see Note 4). For competitive
panning, continue with the following step (see Note 5).
11. Add the following to the selection well and negative control
well: 80 μl of 2Â Blocking Buffer, 20 μl of 80% glycerol and 1 μl
of Halt protease inhibitor cocktail (100Â). If desired, add 1 μg
of non-biotinylated deselection target (see Note 3). Cover with
a plate seal and incubate for 22–24 h at room temperature on
the vibrating platform shaker.
Day 2 or 3
12. Wash the selection well and negative control well 6Â with
300 μl of PBST.
13. Elution 1. Add 100 μl of 0.2 M glycine, pH 2.2 to the selection
well and negative control well. Incubate for 10 min at room
temperature. Neutralize by adding 15 μl of 1 M Tris-HCl,
pH 9.1 and transfer the eluted phage to a
microcentrifuge tube.
14. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Add 100 μl of the diluted triethylamine to the selection well and
negative control well. Incubate for 6 min at room temperature.
Neutralize by adding 50 μl of 1 M Tris-HCl, pH 7 and transfer
the eluted phage to the microcentrifuge tube containing
elution 1.
15. Infection of ER2738 cells. Before eluting the phage, set up
5 ml cultures of ER2738 cells for each selection. Dilute the
overnight cultures of ER2738 cells in 2YT medium with
12 μg/ml tetracycline to an OD600 of ~0.2 and incubate at
37
C, 230 rpm for about 1 h until OD600 reaches ~0.6. Add
the eluted phage and incubate for 1 h at 37
C at up to 90 rpm.
Mix at least once during the incubation.
16. Plating the cells. Plate a range of volumes (e.g., 0.01 μl, 0.1 μl,
1 μl, 10 μl, and 100 μl) onto LB agar plates with 100 μg/ml
carbenicillin. Also, centrifuge and plate the remaining cells as
described for Panning Round 1 (see Subheading 3.1.1, step
13). For the negative control, select one volume to plate
(usually 10 μl). Incubate the plates overnight at 37
C.
Day 3 or 4
17. Compare the number of colonies on the negative control plate
with the selection plate containing the same volume of cells. A
successful screen shows enrichment of target-specific phage
with fewer colonies (less than half) on the negative control
plate. If background is still high on the negative control plate,
Isolation of Artificial Binding Proteins
117