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).
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.
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.
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
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.
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.
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,
A known ligand to the target in TBS (0.1–1 mM)
A free target in TBS (~100 μg/ml)
- 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
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