As a general alternative to panning against a target that has been immobilized on a surface, the library can be reacted with the target in solution, followed by affinity capture of the target–phage complexes onto an affinity matrix (bead) specific for the target protein. For example, if the target protein has a GST, MBP or polyhistidine affinity tag, the target-phage complexes can be captured on glutathione, amylose or chelated nickel beads, respectively. If the target is an antibody, Protein A and/or Protein G beads can be used for capture. In addition to requiring substantially less target per experiment than surface panning, solution panning can result in improved accessibility of the putative ligand binding site to phage-displayed peptides, as well as avoiding partial denaturation of the target on a plastic surface. Fortuitous selection of peptide sequences that specifically bind the bead can be avoided by employing a negative selection beginning with Round 2, in which the amplified phage is pre-incubated with the bead in the absence of target. The supernatant from this step is then reacted with the target in a positive selection. For antibodies or other target proteins that bind to more than one matrix type, bead-specific peptides can be avoided by alternating rounds between the matrix types. For example, for antibody targets, peptides specific for Protein A or Protein G are avoided by alternating rounds of panning between Protein A- and Protein G-agarose (magnetic beads can also be used). For antibodies that do not bind well to Protein A (sheep, goat, chicken and rat polyclonals, as well as some human IgG3 and mouse IgG1 monoclonal antibodies), Protein G-agarose can be used in all rounds, employing a negative selection strategy as described above. Alternatively, Protein A can be used for the mouse IgG1 subclass if the pH of the binding and wash buffer (TBS) is raised to 8.6.
For further information about the NEB phage display protocols and products refer to the PhD manual.
- Inoculate 10 ml of LB+Tet medium with ER2738, for use in titering. If amplifying the eluted phage on the same day, also inoculate 20 ml of LB medium in a 250-ml Erlenmeyer flask (do not use a 50-ml conical tube) with ER2738. Incubate both cultures at 37°C with vigorous shaking. Incubate the titering culture until needed; incubate the 20-ml culture until early-log phase (OD600 0.01–0.05), for use in Step 13.
- Transfer 50 µl of a 50% aqueous suspension of affinity beads appropriate for capture of the target to a microfuge tube. For antibody targets (see above), use Protein A-agarose (or magnetic bead) for the first round if possible. Add 1 ml of TBS + 0.1% Tween (TBST). Suspend the resin by tapping the tube or GENTLY vortexing. Do not pipet up and down.
- Pellet the resin by centrifugation in a low-speed benchtop microcentrifuge for 30 seconds, or by magnetic capture if using magnetic beads. Carefully pipette away and discard the supernatant, taking care not to disturb the resin pellet.
- Suspend the resin in 1 ml of blocking buffer. Incubate for 60 minutes at 4°C, mixing occasionally.
- In the meantime, dilute a 100-fold representation of the library (e.g., 2 x 1011 pfu for a library of complexity 2 x 109) and 2 pmoles of target (= 300 ng for an antibody) to a final volume of 200 μl with TBST. Other buffers (with metal ions etc.) of similar ionic strength can be used if necessary for stabilizing the target. Do not add DTT or other reducing agents if using the Ph.D.-C7C library. The final concentration of target is 10 nM. For low affinity ligands it may be necessary to use target concentrations as high as 1–2 μM (33). Incubate for 20 minutes at room temperature.
- Following the blocking reaction in Step 4, pellet the resin as in Step 3 and wash 4 times with 1 ml of TBST, pelleting the resin each time.
- Transfer the phage–target mixture to the tube containing the washed resin. Mix gently and incubate for 15 minutes at room temperature, mixing occasionally.
- Pellet the resin as in Step 3, discard the supernatant, and wash 10 times with 1 ml of TBST, pelleting the resin each time.
- Elute the bound phage by suspending the resin in 1 ml of Glycine Elution Buffer (0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA) or a solution of a known ligand in TBS (step 10, page 15). Incubate for 10 minutes at room temperature.
- Centrifuge the elution mixture for 1 minute in a low-speed benchtop microcentrifuge or pellet by magnetization. Carefully transfer the supernatant to a new microfuge tube, taking care not to disturb the pelleted resin.
- If eluting with Glycine Elution Buffer, immediately neutralize the eluate with 150 μl of 1 M Tris-HCl, pH 9.1.
- Titer a small aliquot of the eluate on LB/IPTG/Xgal plates as described in General M13 Methods (page 8).
- Amplify the remaining eluate by adding it to the 20 ml ER2738 culture from Step 1 (must be early-log; no later) and incubating at 37°C with vigorous shaking for 4.5 hours.
Alternatively, the eluate can be stored overnight at 4°C and amplified the next day, if preferred. In this case, inoculate 10 ml of LB+Tet with ER2738 and incubate overnight at 37°C with shaking. The next day, dilute the overnight culture 1:100 in 20 ml of LB in a 250-ml Erlenmeyer flask (do not use a 50-ml conical tube) and add the unamplified eluate. Incubate at 37°C with vigorous shaking for 4.5 hours.
- Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
- Pipette the upper 80% of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4°C for 2 hours or overnight.
- Spin the PEG precipitation at 12,000 g rpm for 15 minutes at 4°C. Decant and discard the supernatant, re-spin briefly, and remove the residual supernatant with a pipette.
- Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin for 5 minutes at 4°C to pellet residual cells.
- Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate with 1/6 volume of 20% PEG/2.5 M NaCl. Incubate for 15–60 minutes on ice. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C. Discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
- Suspend the pellet in 200 μl of TBS. Microcentrifuge at 14,000 rpm for 1 minute to pellet any remaining insoluble matter. Transfer the supernatant to a fresh tube. This is the amplified eluate.
- Titer the amplified eluate on LB/IPTG/Xgal plates as described in General M13 Methods, page 8. The eluate can be stored for several weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at -20°C.
- The next day, count blue plaques and determine phage titer (see page 8). Use this value to calculate an input volume corresponding to the input titer used in Step 5. If the titer is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage.
- Perform a second round of panning: repeat Steps 1–21 using the calculated amount of the first round amplified eluate as input phage. Raise the Tween concentration in the binding and wash steps to 0.5% (v/v). For antibody targets, use Protein G-agarose (or magnetic bead) for this round. If using the same resin for all three rounds, a negative selection can be added at this stage if desired (do not carry out a negative selection in the first round):
Prepare an additional 50 μl of washed, blocked resin as in Steps 2–4. Pellet the resin and wash 4 times with TBST. Dilute the library in 200 μl of TBST as in Step 5, but leave out the target. Add the 22 diluted phage to the washed, blocked resin and incubate for 15 minutes at room temperature with occasional mixing. Spin out the resin (or capture magnetically) and transfer the supernatant to a fresh microfuge tube. Add 2 pmoles of target to the supernatant, incubate for 20 minutes at room temperature, and continue with Step 6, using another 50 μl of washed, blocked resin.
- Perform a third round of panning: repeat Steps 1–12 using the calculated amount of the second round amplified eluate as input phage. Keep the Tween concentration at 0.5% (v/v) in the binding and wash steps. For antibody targets, use protein A-agarose (or magnetic bead) if you used protein A for the first round. If using the same resin in all three rounds, a negative selection can be performed as described in Step 22. The eluate can be stored for up to a week at 4°C.
- For preparation of individual clones for sequencing or ELISA, inoculate a 10 ml overnight culture of ER2738 from a colony. Time the titering step so the plates are incubated no longer than 18 hours, as deletions may occur if the plates are incubated longer. Proceed with plaque amplification, page 22. Do not amplify the third round eluate and carry out a fourth round of panning unless the characterization shows no clear consensus sequence or ELISA signal.