Home Protocols Panning Protocol 2: Surface-Phase Panning (Direct Target Coating)

Panning Protocol 2: Surface-Phase Panning (Direct Target Coating)

  • The most straightforward method of affinity partitioning (panning) involves directly coating a plastic surface with the target of interest (by nonspecific hydrophobic and electrostatic interaction), washing away the excess, and passing the pool of phage over the target-coated surface. In this method, the target does not need an affinity tag.
  • Depending on the available quantity of target molecule and the number of different targets being panned against simultaneously, panning can be carried out in individual sterile polystyrene petri dishes, 12- or 24-well plates, or 96-well microtiter plates.
  • Coat a minimum of 1 plate (or individual well) per target. It is not productive to do a separate negative control panning experiment without target.
  • Volumes given in this protocol are for 60 x 15 mm petri dishes, with volumes for microtiter wells given in parentheses. For wells of intermediate size adjust volumes accordingly, but in all cases the number of input phage should remain the same (1011 pfu).

  1. Coating the surface. Prepare a solution of 10–100 μg/ml of the target in 0.1 M NaHCO3, pH 8.6. Alternative buffers (containing metal ions etc.) of similarly high ionic strength (e.g., TBS) can be used if necessary for stabilizing the target molecule. Add 1.5 ml of this solution (150 μl if using microtiter wells) to each plate (or well) and swirl repeatedly until the surface is completely wet (this may take some effort as the solution may bead up). Incubate overnight at 4°C with gentle agitation in a humidified container (e.g., a sealable plastic box lined with damp paper towels). Store plates at 4°C in humidified container until needed. Plates coated with protein targets can usually be stored for several weeks (depending on target stability); discard if mold is evident on the paper towels.

  2. Day culture. Inoculate 10 ml of LB + Tet medium with E. coli K12 ER2738 (NEB #E4104S) for use in tittering. 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 (Panning Protocol 1 Solution-phase Panning with Affinity Bead Capture) but only grown to OD600 0.01-0.05.

  3. Block surface and wash. Pour off the coating solution from each plate and firmly slap it face down onto a clean paper towel to remove residual solution. Fill each plate or well completely with Blocking Buffer. Incubate for at least 1 hour or overnight at 4°C. Then, discard the blocking solution. Wash each plate rapidly 6 times with TBST (TBS + 0.1% [v/v] Tween-20). Coat the bottom and sides of the plate or well by swirling, pour off the solution, and slap the plate face down on a section of dry paper towel each time. (If using a 96-well microtiter plate, an automatic plate washer may be used.) Work quickly to avoid drying out the plate.

  4. Selection Round 1. Dilute 10 μl of the Ph.D. Phage Display Peptide Library (i.e., 1011 pfu, a 100-fold representation of a 109 complexity) with 1 ml of TBST (100 μl, if using microtiter wells). Pipette diluted phage onto coated plate and rock gently for 10–60 minutes at room temperature.

  5. Wash. Discard nonbinding phage by pouring off liquid and slapping plate face-down onto a clean paper towel. Wash plate with 10 x 1 ml (100 μl) TBST.

  6. Elution. Elute the bound phage by applying 1 ml (100 μl, if using microtiter wells) elution buffer to the well or plate. Rock the elution mixture gently for 10–60 minutes at room temperature. Pipette eluate into a microcentrifuge tube.

    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 of selected pool. Go to Panning Protocol 1 Solution-phase Panning with Affinity Bead Capture, Step 7. Choose either Amplification Method A or B. Proceed with amplification and subsequent steps above, 1–6, using the target treated surface.