Using Phage Display to Select Proteins with High Affinity to a Target Protein

1. Prepare the phage library

1. Thaw the phage library on ice and dilute it to 100x the library diversity in PBS. For example, if the library diversity is 1 x 10¹⁰ and the library concentration is 1 x 10¹³, dilute the library so that the concentration is 1 x 10¹². For the libraries used here, dilute them 10-fold in PBS by combining 1 mL of the library with 9 mL of PBS.
   NOTE: Library diversity should have been determined during library creation and cannot be easily assessed otherwise. Library concentration can be determined by inoculating E. coli cells in the mid-log phase (OD600 0.6 – 0.8) and plating on LB/carb.
2. Add 1/5 volume of PEG/NaCl. For example, for 10 mL of the previously diluted library, add 2 mL of PEG/NaCl.
3. Incubate on ice for 30 min.
4. Centrifuge at 11,000 x g for 30 min at 4 °C, discard supernatant, and re-centrifuge for 2 min to pull down the remaining supernatant.
   NOTE: Put the tube in the rotor in the same orientation the second time as it was the first to keep the pellet in the same place, therefore, making it easier to see.
5. Gently resuspend the phage pellet in 1 mL of PBT per protein. For four proteins, resuspend the pellet in 4 mL of PBT.
   NOTE: Try not to touch the pellet and do not introduce air bubbles.

2. Display phage to the target protein(s)

1. Optional control: Add 100 µL of phage library to each coated well in the control plate. Incubate at room temperature (~20-25 °C) for 1 h with 300 rpm orbital shaking and transfer the library from the control plate to the target plate.
2. Remove the PB buffer from the target plate and add 100 µL of the phage library to each coated well.
3. Incubate at room temperature (~20-25 °C) for 1 h with 300 rpm orbital shaking.

3. Elute round one phage

1. Remove the phage library and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops.
   NOTE: If multiple target proteins are coated on one plate, try to minimize the flow of all subsequent solutions between the wells.
2. Add 100 µL of 0.1 M HCl to each coated well and incubate at room temperature for 5 min with 300 rpm orbital shaking.
3. Neutralize the pH by adding 12.5 µL of 1 M Tris-HCl (pH 11) to each coated well.
4. Pool the eluted phage from all 8 wells into a single 1.5 mL microcentrifuge tube. Pipette up and down during the transfer to make solutions homogenous and aspirate all liquid from the wells.
5. Add 10% BSA to the pooled eluted phage to a final concentration of 1%. For a typical round one elution volume of 950 µL, add 95 µL of 10% BSA. Store at 4 °C. This is the round one output.

4. Preparation for subsequent rounds of selection

1. Prepare a seed culture for culturing the phage input for the next round of selection as in step 1.1.
2. Coat the plate for the third round of selection as in step 1.2 with the changes noted below.
   1. Only coat four wells per protein in a 96-well binding plate. Use half of the contents of the "round 2/3" or "round 4/5" tubes for the appropriate round. Dilute the contents of these tubes as needed, in order to avoid proteins settling out of the solution.
3. Prepare the phage input for the next round of selection.
   1. Use half of the round one output to inoculate 3 mL of mid-log phase cells. For a typical round one output, inoculate with 500 µL of the output.
   2. Incubate at 37 °C for 30 min with 200 rpm orbital shaking.
   3. Add M13K07 helper phage to a final concentration of 1 x 10¹⁰ PFU/mL.
   4. Incubate at 37 °C for 1 h with 200 rpm orbital shaking.
   5. Transfer the entire 3 mL of culture to 30 mL of 2YT/carb/kan. Grow overnight at 37 °C with 200 rpm orbital shaking.