Panning Protocol 1: Solution-phase Panning with Affinity Bead Capture
- This protocol can serve as the control panning experiment protocol using DYKDDDDK Mouse mAb target (NEB #E8004) with Protein G Magnetic Beads (S1430).
- This protocol is a recommended starting point for any target that can be pulled out of solution with an affinity tag.
- A straightforward approach is recommended for initial experiments, but for complex or troublesome targets, the current literature for panning protocols in a particular application is invaluable. Refer to Appendix D for discussions on optimizing certain conditions for a given selection.
- Day culture. Inoculate 10 ml of LB+Tet medium with E. coli K12 ER2738 (NEB #E4104S) for use in tittering (Step 10).
Incubate culture at 37°C with vigorous shaking, ~250 RPM, for 4–8 hours.
Culture should be turbid. Consider also starting a culture, if using
Amplification method A (Step 7, part a) but only grow to OD600
- Prepare beads. Transfer 50 μl of a 50% aqueous
suspension of affinity beads appropriate for capture of the target to a
microfuge tube. Add 1 ml of TBS + 0.1% Tween (TBST). Suspend the beads by
tapping the tube or GENTLY vortexing. Do not pipet up and down. Pellet the
beads by magnetic capture if using magnetic beads or by centrifugation in a
low-speed benchtop microcentrifuge for 30 seconds. Carefully pipette away
and discard the supernatant, taking care not to disturb the bead pellet.
Repeat bead wash two more times and set aside to use in Step 4.
Note: A blocking step may be appropriate for some beads. Follow the manufacturer’s instructions if blocking is recommended. Protein G Magnetic Beads (NEB #S1430) are stored in BSA and do not need to be blocked.
- Selection Step 1 (~2 pmol target + phage library).
Dilute 10 μl of the Ph.D. Phage Display Peptide Library (i.e., 10 11 pfu which is a 100-fold representation of a 109
complexity) and 3 μl DYKDDDDK Mouse mAb (NEB #E8004) (if using a different
target, 2 pmol or 300–500 ng) to a final volume of 200 μl with TBST. The
final target concentration is 10 nM. Incubate for 20 minutes at room
temperature with intermittent mixing.
Capture. Transfer the phage–target mixture to the tube
containing the washed beads from Step 2. Incubate for 15 minutes at room
temperature, mixing occasionally.
Wash. Pellet the beads as in Step 2, discard the
supernatant, and wash 10 times with 1 ml of TBST, pelleting the beads each
time. Discard wash volume.
Elution. Elute the bound phage by suspending the beads
in 1 ml of elution buffer. Intermittently mix for 10–60 min. For the
control panning experiment, use Glycine Elution Buffer (see below).
Possible Elution Buffers:
- 0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA; must be neutralized afterwards by adding 150 μl (15 μl for microtiter wells) of sterile 1 M Tris-HCl, pH 9
- A known ligand to the target in TBS (0.1–1 mM)
A free target in TBS (~100 μg/ml)
- Amplification Method A: Inoculate 20 ml E. coli K12 ER2738 LB culture (from Step 1) with a small mass of cells or single colony. Shake flask at 250 RPM at 37°C for an hour or less to achieve OD 600 0.01–0.05. The culture will not be turbid. Take care not to over grow. Add phage eluate to the culture within the recommended low OD 600 range, continue incubation with vigorous shaking for 4.5–5 hours.
- Amplification Method B: Amplify eluate the next day. Inoculate 10
ml of LB + Tet with E. coli K12 ER2738 (NEB# E4104S) and incubate
overnight at 37°C with shaking. The next day, dilute the overnight culture
1:100 in 20 ml of LB and at the same time add the unamplified eluate.
Incubate at 37°C with vigorous shaking for 4.5 hours. This approach does
not call for monitoring OD600.