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Phage Capture Assay: Micropanning
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
Two main binding assays are used to confirm the specific binding of target receptors (ligate) to phage-borne ligands: ELISA (see Protocol ID#2162) and the phage capture assay (micropanning) described in this protocol. ELISA has the advantage that it confirms that the ligate binds the phage by a different method from the affinity selection procedure (see Protocol ID#2179) used originally to obtain the phage. However, ELISA is not very sensitive: even phage that have been successfully and specifically affinity-selected sometimes give a feeble ELISA signal that is indistinguishable from background. In contrast, the phage capture assay is far more sensitive than ELISA.
Procedure
1. Pipette 40 μl of Streptavidin Solution into each well of a 96-well polystyrene ELISA plate (see Hint #2).
2. Allow the protein to adsorb to the plastic for at least 1 hr at room temperature.
3. Aspirate the Streptavidin Solution and fill the wells to the brim with 400 μl of Blocking Solution per well.
4. Allow the ELISA plates to sit at room temperature for 2 hr with the lids removed.
5. Wash the ELISA plates five times with TBS/Tween, preferably on an automated plate washer.
6. Add the desired amount of biotinylated ligate (see Protocol ID#2188; see Hint #3) in 200 μl of TTDBA.
7. Leave the plate to react for at least 2 hr at 4°C in a humidified box.
8. Wash the plate five times with TBS/Tween to remove any unbound receptor, and fill the wells with 100 μl of TTDBA.
9. Add 30 μl of input phage (see Protocol ID#2169; see Hint #4 and Hint #5).
10. Rock the plate for 4 hr at 4°C.
11. Let the reaction continue for 2 hr at room temperature in a humidified box.
12. Wash the wells ten times with TBS/Tween.
13. Into each well pipette 20 μl of Elution Buffer (containing Phenol Red). Pipette 20 μl of Elution Buffer into a few blank wells also, to serve as mock eluates.
14. Let sit for approximately 10 min at room temperature.
15. Pipette the eluate (or mock eluate) in each well into the corresponding well of a second microtiter plate (see Hint #6) to which 3.75 μl of 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 (see Hint #7).
16. Using up to 4 additional microtiter plates, (see Hint #6) prepare serial 10-fold dilutions by passing 2.2 μl of the neutralized eluate or previous dilution into 20 μl of TBS/Gelatin as the diluent (See Protocol ID#2181).
17. To get an accurate percent yield, it is necessary to titer the input as well as the output phage. Pipette 20 μl of a suitable dilution (see Hint #8) of the input virions (see Step #9) into other wells.
18. Pipette 20 μl of starved K91BluKan cells into each well in the plates from Step #15 to Step #17 (see Protocol ID#2173).
19. Allow the infection to proceed at room temperature for 10 min.
20. Pipette 200 μl of NZY Media containing Tetracycline into each well.
21. Place the microtiter plates in the 37°C incubator for approximately 30 min (see Hint #9).
22. Spot a 20 μl portion of each well onto an NZY plate containing Tetracycline and Kanamycin (see Hint #10). If the plates have been dried in advance, a single standard 100 mm petri-plate can accommodate 19 spots in a hexagonal array (see Image #1).
23. Count the colonies in the spots after approximately 12 hr at 37°C (see Hint #11).
Solutions
Elution buffer
0.1 M HCl, pH adjusted to 2.2 with Glycine (see Hint #1)
1 mg/ml BSA
0.1 mg/ml Phenol Red 
TTDBA
Prepare in TBS/Tween
1 mg/ml BSA
0.02% NaN3
TBS/Tween
Prepare in TBS
0.5 % (v/v) Tween-20
TBS/Gelatin
Store at room temperature
Autoclave 0.1 g Gelatin in 100 ml TBS
After autoclaving, swirl to mix in the melted gelatin
TBS (1X)
50 mM Tris HCl, pH 7.5
Store at room temperature
Autoclave
150 mM NaCl
Blocking Solution
0.1 M NaHCO3
0.1 μg/ml Streptavidin
5 mg/ml dialyzed Bovine Serum Albumin (BSA)
0.02% NaN3
Streptavidin Solution
10 μg/ml Streptavidin
Prepare in 0.1 M NaHCO3
NZY plates containing Tetracycline and Kanamycin
The Media is now approximately 60°C.
Dry overnight at room temperature or for a few hours in the 37°C incubator before use (see Hint #14)
Pour into plates (do not allow the temperature to fall below 50°C), adding sufficient media to cover the bottom of the plates.
Add 500 ml of 2X liquid Media (NZY) (see Hint #13)
Add 100 μg/ml Kanamycin.
While the agar is autoclaving, set out and label empty petri plates
Add 11 g of Bacto agar
Add 40 μg/ml Tetracycline
Measure 500 ml of ddH2O into a 2 liter polypropylene Erlenmeyer flask (see Hint #12)
Cover with a polypropylene or glass beaker and autoclave.
Allow the plates to cool to room temperature.
Plates
The Media is now approximately 60°C. Antibiotics can be added at this temperature.
Dry overnight at room temperature or for a few hours in the 37°C incubator before use (see Hint #14)
Add 500 ml of 2X liquid Media (NZY or LB) (see Hint #13)
Pour into plates (do not allow the temperature to fall below 50°C), adding at least enough to cover the bottom.
While the agar is autoclaving, set out and label empty petri plates
Add 11 g of Bacto agar
1 liter (see Hint #11)
Measure 500 ml of ddH2O into a 2 liter polypropylene Erlenmeyer flask (see Hint #12)
Cover with a polypropylene or glass beaker and autoclave.
Allow the plates to cool to room temperature.
NZY Media containing Tetracycline
Adjust pH to 7.5 with NaOH
5 g NaCl
Autoclave and store at room temperature
Dissolve in 1 liter of ddH2O
Add 0.2 μg/ml Tetracycline
5 g Yeast Extract
10 g NZ Amine A
BioReagents and Chemicals
Sodium Azide
Bovine Serum Albumin
Hydrochloric Acid
Sodium Hydroxide
Yeast Extract
Sodium Bicarbonate
Gelatin
NZ Amine A
Glycine
Tetracycline
Streptavidin
Phenol Red
Tween-20
Bacto Agar
Kanamycin
Tris HCl
Sodium Chloride
Protocol Hints
1. The Elution Buffer is prepared and adjusted as a 4X stock, filter-sterilized, and stored at room temperature. The phenol red gives a visual indication of whether the pH is (roughly) correct after the eluate is neutralized.
2. The contributors use a modified flat-bottom plate that fits in the automated plate washer.
3. Typically 1 to 500 ng are used. 500 ng is more than sufficient to saturate the immobilized streptavidin).
4. The physical particle concentration of the input phage can be up to approximately 1010 virions/ml (approximately 5 X 108 Transducing Units/ml; see Protocol ID#2173). 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. Potential blocking inhibitors can be added before the virions if desired to check the specificity of binding or for other reasons.
5. Any of three methods for propagating virions in the Protocol ID#2169 may be used for this protocol. The contributors suggest that either double PEG-precipitated virions (Section B) or double PEG-precipitated, acid-precipitated virions (Section C) be used. Another alternative is using virions propagated according to Protocol ID#2177 and Protocol ID#2178. Other antigens, such as purified or recombinant protein, can be used as the immobilized species when appropriate.
6. Inexpensive flexible microtiter plates are acceptable at this step.
7. Avoid neutralizing the eluate in the original wells. In many cases the phage can re-bind the immobilized ligate after neutralization.
8. Use TBS/Gelatin as the diluent. The desired concentration is 105 physical particles/ml.
9. 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.
10. The input dilutions with 105 virions/ml should generate approximately 10 colonies on the spot. Background yield (theoretically approximately 3 X 10-5% input) corresponds to less than 1 colony on the control spot, but typically we find approximately 10. High specific yields (approximately 10%) correspond to approximately 10 colonies on the 10-4spot.
11. This is the recipe for 1 liter, enough for approximately 40 plates; scale up or down as appropriate. Two-fold concentrated (2X) liquid medium is prepared and autoclaved in advance, and stored at room temperature in polypropylene bottles (The contributors of this protocol find it easier to pour from polypropylene than glass while keeping the solution sterile because of the tendency toward "drip-back" occurring with the use of glass).
12. Use a flask that has at least twice the capacity of the final volume of the solution. Again, plastic is preferable to glass.
13. Pour the medium gently down the wall of the flask, which should be held at an angle to prevent the medium from splashing down directly into the agar. These manipulations are designed to minimize the formation of bubbles, which are exceedingly hard to remove. Mix the contents of the flask by gentle rotation, holding the flask at a shallow angle to promote mixing. Again, avoid bubbles.
14. Plates can be dried within a few hours upside-down tilted out of their lids in a sterile laminar flow hood.
