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.

1. 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 0.01–0.05.
   
   
2. 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.
   
   
3. 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.
   
   
4. 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.
   
   
5. 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.
   
   
6. 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)
  
  
7. Enrichment (amplification) of selected pool. Retain a 5-10 μl volume of the eluate, immediately amplify the remaining eluate or store at 4°C for up to 2 weeks. Later rounds (with higher titers) may be stored at -20°C indefinitely with the addition of an equal volume of glycerol. Amplify the phage pool in a 250 ml Erlenmeyer flask (do not use a 50 ml conical tube) and culture by either method below:
   
   
   1. 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.
   2. 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.
      
      
8. Remove cells. Transfer the culture to a centrifuge tube and spin at 4°C for 10 minutes at 5,000 x g. Transfer the supernatant to a fresh tube and briefly re-spin.
   
   
9. Precipitate phage. Pipette 16 ml of the culture supernatant to a fresh tube and add to it 4 ml (or 1/5 volume) of 20% PEG/2.5 M NaCl in a centrifuge tube. Mix gently and allow the phage to precipitate at 4°C for 2 hours or overnight. Spin the PEG precipitation at 12,000 x g for 15 minutes at 4°C (alternatively, 4,000 x g for 45 minutes). A fingerprint sized smear of phage pellet may be visible. Decant the supernatant. (If a phage pellet is not visible, save the supernatant at 4°C; the culture supernatant may be tittered later if troubleshooting of the amplification steps is necessary.) Add 1 ml of TBS to the phage pellet. Mix by gentle pipetting or vortex several times over 5 minutes. Transfer the suspension to a microcentrifuge tube and spin for 1 minute at maximum speed (~20,000 x g) at 4°C to pellet residual cells. Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate with 200 μl of 20% PEG/2.5 M NaCl. Incubate for 15– 60 minutes on ice. Solution will rapidly appear cloudy. Microcentrifuge for 10 minutes at 20,000 x g at 4°C. Discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipette. Suspend the pellet in 200 μl of TBS. Microcentrifuge at max speed for 1 minute to pellet any remaining insoluble matter. Transfer the supernatant to a fresh tube. This is the amplified eluate.
   
   
10. Determine pfu/μl of the amplified eluate by titration. Titer the amplified eluate as described in the protocol Phage Titering. Typically, the titer will be on the order of 10¹⁰ pfu/μl (or 10 ¹³ pfu/ml) for an amplified, PEG/NaCl concentrated stock.
    
    
11. Selection round 2. Using 10¹¹ pfu of the amplified eluate from the previous round, perform a second round of selection by repeating Steps 1–7, raising the Tween concentration in the binding and wash steps to 0.5% (v/v). If the amplified eluate titer is low, succeeding rounds of panning can be carried out with as little as 10 ⁹ pfu of input phage, if need be.
    
    
12. Additional selection steps. If needed, perform a third round of selection as above maintaining the Tween concentration at 0.5% (v/v) in the binding and wash steps. Store the eluate at 4°C.
    
    
13. Do not immediately amplify the third-round eluate. Instead, obtain DNA for sequencing reactions from 10–20 clones (see Post Panning Protocol 1: Rapid Purification of Single-Stranded DNA Templates for Sequencing Reactions). Also, consider binding assays with synthetic peptides or whole phage ELISA (see Phage ELISA Binding Assay with Direct Target Coating) to interrogate specificity for the target.