Phage Capture Assay ( Micropanning )

0df3c390d51fa5128d4e8678f43d75fb.doc 04/05/18 Page 1

Phage Capture Assay (“Micropanning”)

NOTE: We use two main binding assays to confirm specific binding of ligate to phage-borne ligands: ELISA (ELISA.doc) and the phage capture assay (alias “micropanning”) described in this document. ELISA has the advantage that it confirms that the ligate binds the phage by a completely different method from the affinity selection procedure (AffinitySelection.doc) used to obtain the phage in the first place; it also lends itself to semi-quantitative analysis of binding constants. On the other hand, ELISA is not very sensitive: even phage that have been successfully and specifically affinity-selected sometimes give a feeble ELISA signal that is indistiguishable from background, which probably reflects predominantly reaction of ligate with non-phage impurities. In contrast, the phage capture assay—a miniaturized version of “one-step” affinity selection applied to individual clones rather than whole libraries or selected subpopulations—is far more sensitive than ELISA. Moreover, since the signal it measures is titer of infective phage particles, there is no non-phage background to obscure the signal.

NOTE: We assume here that an automated plate washer is available; if not, the wells have to be washed by hand with a squirt bottle.

1. Into each well of a 96-well polystyrene ELISA dish (we use a modified flat-bottom dish that fits in our plate washer) pipette 40 µl 10 µg/ml streptavidin in 0.1 M NaHCO3; allow the protein to adsorb to the plastic for at least 1 hr at room temperature.

2. Aspirate out the streptavidin solution and fill the the wells to brimming with blocking solution (~400 µl/well); allow to sit with lid off for 2 hr at room temperature.

3. Wash the dish five times with TBS/Tween, preferably on an automated plate washer.

4. Add the desired amount of biotinylated ligate (typically 1–500 ng, the latter being more than enough to saturate the immobilized streptavidin) in 200 µl TTDBA; allow the dish to react at least 2 hr at 4º in a humidified box.

5. Wash the dish five times with TBS/Tween as in step 3 to remove unbound receptor, and fill the wells with 100 µl of TTDBA.

6. Add 30 µl input phage (see note below) and rock the dish 4 hr (usually at 4°, but sometimes at other temperatures). React for ~2 hr at room temperature in a humidified box.

NOTE: The physical particle concentration of the input phage can be up to ~1010 virions/ml (~5 × 108 TU/ml); for example, this might be a 1/50 dilution of a culture supernatant in TTDBA diluent. If only tight-binding clones are of interest, the input virion concentration can be reduced below this upper limit in order to reduce the number of dilutions to be titered. If it is desired only to ascertain qualitatively whether or not a clone binds strongly to the ligate, use an input physical particle concentration of ~107 virions/ml, and skip the dilutions at step 10 and the input titer at step 11 (see note after the final step below). Potential blocking inhibitors can be added before the virions if desired to check the specificity of binding or for other reasons.

7. Wash the wells ten times with TBS/Tween (step 3). 0df3c390d51fa5128d4e8678f43d75fb.doc 04/05/18 Page 2

8. Into each well pipette 20 l elution buffer (with phenol red). Also pipette 20 l elution buffer into a few blank wells to serve as mock eluates. Allow dish to sit ~10 min at room temperature.

9. Pipette the eluate (or mock eluate) in each well into the corresponding well of a second microtiter dish (a cheap flexible microtiter dish is OK) to which 3.75 l 1 M Tris.HCl pH 9.1 has already been added. Confirm that the phenol red turns from yellow to red, indicating successful neutralization of the acid in the elution buffer.

NOTE: It is not a good idea to neutralize the eluate in the original wells, since in many cases the phage can re-bind the immobilized ligate after neutralization!

10. In up to 4 additional microtiter dishes (again, cheap flexible microtiter dishes are OK) make serial 10-fold dilutions by passing 2.2 l of the neutralized eluate or previous dilution into 20 l TBS/gelatin diluent.

11. If it is desired to titer the input as well as the output phage to get an accurate percent yield, you should also pipette 20 l of a suitable dilution (diluent = TBS/gelatin) of the input virions (step 6; aim at 105 physical particles/ml) into other wells.

12. Into each well in the plates from steps 9–11 pipette 20 l of starved K91BluKan cells (TUtiter.doc, steps 1–7). Allow infection to proceed at room temperature for 10 min.

13. Into each well pipette 200 l NZY containing 0.2 g/ml tetracycline; put microtiter dishes in the 37° incubator for ~30 min. (This is the gene expression period, during which the tetracycline resistance gene on the incoming phage has a chance to be expressed before the infected cells are challenged with a high concentration of the antibiotic.)

14. Spot a 20-l portion of each well onto an NZY plate containing 40 g/ml tetracycline and 100 g/ml kanamycin. If the plates have been dried in advance, a single standard 100-mm petri dish can accommodate 19 spots in a hexagonal array.

15. Count the colonies in the spots after ~12 hr at 37°.

NOTE: The input dilutions with 105 virions/ml should give ~10 colonies on the spot. Background yield (theoretically ~3 × 10-5% input) corresponds to less than 1 colony on the neat spot, but typically we get ~10. High specific yields (~10%) correspond to ~10 colonies on the 10-4 spot. If it is desired simply to determine qualitatively whether each clone binds specifically or not, use input virions at ~107 virions/ml as the input at step 6, and skip the dilutions at step 10; 0df3c390d51fa5128d4e8678f43d75fb.doc 04/05/18 Page 3 in these circumstances, excellent binding (10% yield) corresponds to ~100 colonies/spot, good binding (~1% yield) to ~10 colonies/spot), while background should be 0.