11. Resuspend the washed beads in the following: 77 μl of PBS,
60 μl of 10Â Blocking Buffer, 60 μl of 80% glycerol, and 3 μl of
Halt protease inhibitor cocktail (100Â). If desired, add 1 μg of
non-biotinylated deselection target (see Note 3). Incubate for
22–24 h at room temperature on the tube rotator.
Day 2 or 3
12. Centrifuge the tube at 800 Â g for 1 min. Wash the beads 6Â
with 500 μl of 2Â Blocking Buffer using the magnetic separation rack to immobilize the beads.
13. Elution 1. Resuspend the beads in 100 μl of 0.2 M glycine,
pH 2.2 to elute the phage. Incubate for 10 min at room
temperature, placing back on the magnet for the last minute.
Transfer the eluted phage to fresh tubes containing 15 μl of
1 M Tris-HCl, pH 9.1 to neutralize.
14. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Elute any remaining phage by resuspending the beads in 100 μl
of the diluted triethylamine. Incubate for 6 min at room temperature, placing back on the magnet for the last minute.
Transfer the eluted phage to fresh tubes containing 50 μl of
1 M Tris-HCl, pH 7 to neutralize. Pool with the eluted phage
from Elution 1.
15. Infection of ER2738 cells. Before eluting the phage, set up an
8 ml culture 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. Centrifuge the phage-infected culture at
3000 Â g for 5 min. Pour off the supernatant and resuspend
the cell pellet in the residual supernatant. Plate the cells on LB
agar with 100 μg/ml carbenicillin. Incubate the plates overnight at 37
C.
Day 3 or 4.
17. Propagate the phage from Panning Round 2 as described for
Panning Round 1 (see Subheading 3.1.1, steps 15–22).
3.1.3 Panning Round 3
Day 1
1. Block the NeutrAvidin-coated wells with 300 μl per well of
2Â Blocking Buffer. Prepare six wells per selection—four wells
for pre-panning the phage, one for panning against the immobilized target, and a negative control well for panning against
either a deselection target or an empty well. Cover the wells
with an adhesive plate seal and incubate overnight at 37
C.
Isolation of Artificial Binding Proteins
115
60 μl of 10Â Blocking Buffer, 60 μl of 80% glycerol, and 3 μl of
Halt protease inhibitor cocktail (100Â). If desired, add 1 μg of
non-biotinylated deselection target (see Note 3). Incubate for
22–24 h at room temperature on the tube rotator.
Day 2 or 3
12. Centrifuge the tube at 800 Â g for 1 min. Wash the beads 6Â
with 500 μl of 2Â Blocking Buffer using the magnetic separation rack to immobilize the beads.
13. Elution 1. Resuspend the beads in 100 μl of 0.2 M glycine,
pH 2.2 to elute the phage. Incubate for 10 min at room
temperature, placing back on the magnet for the last minute.
Transfer the eluted phage to fresh tubes containing 15 μl of
1 M Tris-HCl, pH 9.1 to neutralize.
14. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Elute any remaining phage by resuspending the beads in 100 μl
of the diluted triethylamine. Incubate for 6 min at room temperature, placing back on the magnet for the last minute.
Transfer the eluted phage to fresh tubes containing 50 μl of
1 M Tris-HCl, pH 7 to neutralize. Pool with the eluted phage
from Elution 1.
15. Infection of ER2738 cells. Before eluting the phage, set up an
8 ml culture 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. Centrifuge the phage-infected culture at
3000 Â g for 5 min. Pour off the supernatant and resuspend
the cell pellet in the residual supernatant. Plate the cells on LB
agar with 100 μg/ml carbenicillin. Incubate the plates overnight at 37
C.
Day 3 or 4.
17. Propagate the phage from Panning Round 2 as described for
Panning Round 1 (see Subheading 3.1.1, steps 15–22).
3.1.3 Panning Round 3
Day 1
1. Block the NeutrAvidin-coated wells with 300 μl per well of
2Â Blocking Buffer. Prepare six wells per selection—four wells
for pre-panning the phage, one for panning against the immobilized target, and a negative control well for panning against
either a deselection target or an empty well. Cover the wells
with an adhesive plate seal and incubate overnight at 37
C.
Isolation of Artificial Binding Proteins
115