Day 2
3. Centrifuge the blocked streptavidin beads at 800 Â g for 1 min.
Place the tube on the magnetic separation rack to immobilize
the beads. Remove the blocking buffer and replace with fresh
2Â Blocking Buffer, resuspending the beads in 100 μl per 20 μl
of streptavidin beads.
4. Immobilization of biotinylated target. In an Eppendorf
LoBind microcentrifuge tube, add 1 μg of biotinylated target
to 200 μl of 2Â Blocking Buffer and 50 μl of the blocked
streptavidin beads. Incubate at room temperature on a tube
rotator to keep the beads in suspension.
5. Immobilization of biotinylated deselection target (if using) for
pre-panning phage (see Note 1). Add 1 μg of biotinylated
deselection target to 200 μl of 2Â Blocking Buffer and 50 μl
of the blocked streptavidin beads. Incubate for 1 h at room
temperature on the tube rotator. 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. Resuspend the beads in 50 μl of 2Â Blocking Buffer.
6. Pre-pan 1. In an Eppendorf LoBind microcentrifuge tube, add
125 μl of phage-containing supernatant from panning round
1 to 125 μl of 2Â Blocking Buffer and 25 μl of the blocked
streptavidin beads (from step 3 or step 5, as appropriate).
Incubate for 1 h at room temperature on the tube rotator.
7. Pre-pan 2. Centrifuge the pre-pan 1 tube at 800 Â g for 1 min
and place the tube on the magnetic separation rack to immobilize the beads. Transfer the phage-containing supernatant to
the tube containing the remaining 25 μl of blocked streptavidin
beads (from step 3 or step 5, as appropriate). Incubate for 1 h
at room temperature on the tube rotator.
8. Centrifuge the tube containing the immobilized target 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.
9. Selection. Centrifuge the pre-pan 2 tube at 800 Â g for 1 min
and place the tube on the magnetic separation rack to immobilize the beads. Transfer the phage-containing supernatant to
the tube containing the immobilized target. If desired, add
1 μg of non-biotinylated deselection target (see Note 3). Incubate for 1 h at room temperature on the tube rotator.
10. 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. 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).
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3. Centrifuge the blocked streptavidin beads at 800 Â g for 1 min.
Place the tube on the magnetic separation rack to immobilize
the beads. Remove the blocking buffer and replace with fresh
2Â Blocking Buffer, resuspending the beads in 100 μl per 20 μl
of streptavidin beads.
4. Immobilization of biotinylated target. In an Eppendorf
LoBind microcentrifuge tube, add 1 μg of biotinylated target
to 200 μl of 2Â Blocking Buffer and 50 μl of the blocked
streptavidin beads. Incubate at room temperature on a tube
rotator to keep the beads in suspension.
5. Immobilization of biotinylated deselection target (if using) for
pre-panning phage (see Note 1). Add 1 μg of biotinylated
deselection target to 200 μl of 2Â Blocking Buffer and 50 μl
of the blocked streptavidin beads. Incubate for 1 h at room
temperature on the tube rotator. 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. Resuspend the beads in 50 μl of 2Â Blocking Buffer.
6. Pre-pan 1. In an Eppendorf LoBind microcentrifuge tube, add
125 μl of phage-containing supernatant from panning round
1 to 125 μl of 2Â Blocking Buffer and 25 μl of the blocked
streptavidin beads (from step 3 or step 5, as appropriate).
Incubate for 1 h at room temperature on the tube rotator.
7. Pre-pan 2. Centrifuge the pre-pan 1 tube at 800 Â g for 1 min
and place the tube on the magnetic separation rack to immobilize the beads. Transfer the phage-containing supernatant to
the tube containing the remaining 25 μl of blocked streptavidin
beads (from step 3 or step 5, as appropriate). Incubate for 1 h
at room temperature on the tube rotator.
8. Centrifuge the tube containing the immobilized target 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.
9. Selection. Centrifuge the pre-pan 2 tube at 800 Â g for 1 min
and place the tube on the magnetic separation rack to immobilize the beads. Transfer the phage-containing supernatant to
the tube containing the immobilized target. If desired, add
1 μg of non-biotinylated deselection target (see Note 3). Incubate for 1 h at room temperature on the tube rotator.
10. 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. 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).
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Anna A. S. Tang et al.