Biopanning of Phage Displayed Peptide Libraries
for the Isolation of Cell-Specific Ligands
Michael J. McGuire, Shunzi Li, and Kathlynn C. Brown
One limitation in the development of biosensors for the early detection of disease is the availability of
high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process. Within
the past 10 years, biopanning of phage displayed peptide libraries on intact cells has proven to be a suc-
cessful route to the identification of cell-specific ligands. The peptides selected from these combinatorial
libraries are often able to distinguish between diseased cells and their normal counterparts as well as
cells in different activation states. These ligands are small and chemical methodologies are available for
regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and
therapeutic platforms. Here we describe the methods utilized in the selection of peptides from phage dis-
played libraries by biopanning. In addition, we provide methods for the synthesis of the selected peptides
as both monomers and tetramers. Downstream uses for the peptides are illustrated.
Key words: Phage display, Peptides, Cell-targeting, Biopanning, Combinatorial library, Diagnostics,
Therapeutics, Quantum dots.
The development of biosensors for the detection of different dis-
ease states is dependent on the availability of high affinity and
specificity ligands for the desired cell type and/or biomarker. In
many applications, the accessibility of such ligands has been the
limiting factor in the development of the technology. To date,
antibodies have been the most common class of ligands utilized.
However, antibodies are expensive and can be difficult to modify.
Additionally, if the down-stream application is to detect particu-
lar cell types (i.e., a cancerous cell vs. its normal counterpart), the
antibody must bind to its target in the context of an intact cell.
Avraham Rasooly and Keith E. Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol. 504
© Humana Press, a part of Springer Science + Business Media, LLC 2009
292 McGuire, Li, and Brown
As such, our lab and others have turned toward peptide librar-
ies as a source of cell-specific ligands (1–16). In the same fashion
that phage displayed peptide libraries can be panned on purified
biomolecules, whole cells can be used as the bait for the peptide
library. This approach, often referred to as biopanning, results in
the isolation of peptides that display high cell-specificity; ligands
can be isolated that discriminate between cell types and disease
states. Furthermore, cell-specific peptides can be obtained with-
out the knowledge of a suitable cell surface biomarker. The pro-
tocol is amenable to a variety of different cell types, including
primary cells. To date, we have identified cell-specific peptides
for many different cell types including cells of the immune sys-
tem (2, 4), pancreatic β-cells (7), cardiac cells (3), tumor cells (5,
6), and pathogen-infected cells (8). Importantly, most peptides
selected in this manner are active outside of context of the phage,
retaining their cell-specificity and affinity. Furthermore, we have
shown that tetramerizing the peptides on a branched scaffold
can greatly enhance the peptides affinity for its target cell type
(5, 6, 8, 17, 18). These peptides can be employed for the deliv-
ery of fluorescent nanoparticles, as cell capture reagents for cell
enrichment, and as antibody replacements for flow cytometry. As
peptides are amenable to derivatization, we anticipate that these
cell-specific ligands will find utility in a variety of different biosen-
1. Tissue culture cell line or primary cell of interest.
2. Tissue culture plates (12-well) for adherent cells.
3. Polypropylene centrifuge tubes (15 mL and 50 mL) for non-
4. Microcentrifuge tubes (1.5 mL).
5. Cell scrapers.
6. Phage library (see Note 1) or amplification stock for each
round of panning.
7. RPMI media (or any cell-specific media) without serum.
8. Chloroquine stock (100×): Dissolve 55 mg chloroquine in
10 mL PBS for a final concentration of 10 mM. Filter steri-
lize the solution.
9. Protease inhibitor (25×) without EDTA (Roche).
10. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4.
2.1. Cell Culture
and Phage Panning
(Methods Outlined in
Biopanning of Phage Displayed Peptide Libraries 293
11. PBS+: Add 0.5 mM CaCl2 and 10 mM MgCl2 to PBS in this
order with stirring.
12. PBS+ with 0.1% BSA: Add 0.1 g bovine serum albumin per
100 mL PBS+.
13. 0.1 M HCl–glycine, 0.9% NaCl pH adjusted to 2.2 with
14. 1.5 M Tris–HCl, pH 8.8.
15. 30 mM Tris–HCl, pH 8.0.
1. Selective media for K91 bacterial stocks. We use M9-Pro
minimal medium prepared as follows: Mix 7.5 g agar +
430 mL water and autoclave solution. Cool agar solution to
∼55°C. Add 25 mL 20× M9 Salts, 5 mL 20% glucose, 50 μL
1 M CaCl2, 500 μL 1 M MgSO4, 100 μL 0.1% Thiamine,
2.5 mL 0.2 mg/mL biotin, 2.5 mL 1% uridine, 8 mL 1% leu-
cine, 8 mL 1% phenylalanine, 8 mL 1% threonine, 8 mL 1%
methionine, 8 mL 1% histidine, 8 mL 1% Tryptophan, and
8 mL 1% lysine. 20× M9 salts consist of 60 g Na2HPO4, 30 g
KH2PO4, 5 g NaCl, and 10 g NH4Cl.
2. LB media.
3. 100 mm and 150 mm LB-tet plates (12 μg/mL tetracycline).
4. Culture flasks for expansion and isolation of phage clones.
5. 20% PEG-8000 (Fisher Chemical) in 0.9% NaCl (see Note 2).
6. 65°C heating block.
7. Bacterial incubator with shaker.
8. Various centrifuge tubes and bottles.
9. Low (3,000 × g) and high speed (11,000 × g) centrifuges for
concentrating bacterial stocks and phage isolation.
10. Spectrophotometer to monitor bacterial cultures.
11. PBS prepared as described in Subheading 2.1.
1. BioRad iCycler or similar apparatus.
2. 2× Sybr® green mastermix (see Note 3).
3. Optical PCR plates for real-time PCR.
4. Optical sealing tape for real-time PCR.
5. 8-channel pipette (5–50 μL).
6. Serial dilutions of previously characterized phage preparation
to generate a standard curve.
7. Specific primers to tetracycline resistance gene (see Note 4)
(a) Forward primer (tetR-F1): 5′-CGAATAAGAAGGCTGG
(b) Reverse primer (tetR-R1): 5′-GCTGTGGGGCATTTTAC
2.2. Bacterial Culture
and Phage Amplifi-
cation and Titering
(Methods Outlined in
Real-Time PCR for
Titering (Method Out-
lined in Subheading
294 McGuire, Li, and Brown
1. General materials for PCR mastermix preparation: 10×
polymerase buffer, 25 mM MgCl2, 10 mM dNTP mix, Taq
polymerase (GoTaq® DNA Polymerase 5 units/μL, Promega
Corp, or Choice™ Taq, Denville Scientific).
3. Specific primers that flank library site
(a) Forward primer (fd-tet F1): 5′-GGGCGATGGTTGTTGT
(b) Reverse primer (fd-tet B1): 5′-CTCATTTTCAGGGATAG
4. Agarose gel apparatus.
5. 100 bp ladder standards (Promega Corp, catalog # G2101 or
6. Exonuclease I (10 units/μL, New England Biolabs, or other
7. Shrimp alkaline phosphatase (1 unit/μL, New England
Biolabs or other suitable vendor).
8. BigDye® Terminator v3.1 (Applied Biosystems Inc).
9. Ethanol (70%).
10. Hi-Di™ Formamide (Applied Biosystems Inc).
11. Sequencing stop/precipitation reagent: Prepare by mixing
125 mL 95% ethanol, 29 mL water, and 6 mL 3 M sodium
acetate, pH 5.2.
1. Materials outlined in Subheading 2.1 for the panning and
Subheading 2.2 or Subheading 2.3 for titering.
2. Isolated phage clones and a control phage clone displaying
an irrelevant peptide sequence. Alternatively, a phage clone
that displays no peptide (referred to as an “empty” phage)
can be employed.
3. Cell lines or primary cells of interest.
1. Symphony Synthesizer (Rainin Instruments, Protein Tech-
nologies, Inc. Woburn, MA) or other standard solid phase
2. Resins for solid phase synthesis: Rink Amide AM resin (substi-
tution level 0.71 mmol/g, Novabiochem, EMD Biosciences,
San Diego, CA); Fmoc4-Lys2-Lys-β-Ala-CLEAR™ Acid
Acid Resin and Fmoc4-Lys2-Lys-Cys(Acm)-β-Ala-CLEAR™
Acid Resin (substitution level 0.21 mmol/g, Peptides Inter-
national, Louisville, KY).
3. Fmoc amino acids required to synthesize desired peptide. Pre-
pare 200 mM amino acid solutions by dissolving 20 mmol Fmoc-
protected amino acids in DMF to final volume of 100 mL.
2.4. Colony PCR for
nation (Outlined in
2.5. Selectivity and
Synthesis (Outlined in
Biopanning of Phage Displayed Peptide Libraries 295
4. Coupling reagents: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-te-
tramethyluronium hexafluorophosphate (HBTU), 1-Hy-
droxybenzotrizole (HOBt) all available from Novabiochem.
Prepare 200 mM solutions as follows: Weigh out 18.965 g
HBTU,6.755 g HOBt, and 11 mL NMM, Add DMF to final
volume of 250 mL.
5. If desired nonnatural amino acids can be incorporated into the
peptide. We routinely incorporate Fmoc-NH–(PEG)11–COOH
(C42H65NO16) (Polypure, Oslo, Norway), Fmoc-Glu(biotinyl-
PEG)–OH (C40H55N5O10S), and Fmoc-Lys(biotin)–OH
(C31H38N4O6S) (Novabiochem, EMD Biosciences, San Diego,
CA) (see Notes 5 and 6).
6. Piperidine in DMF (20%): 200 mL piperidine, 800 mL
7. Cleavage cocktails (see Note 7):
(a) TFA: H2O:TIS (95%:2.5%:2.5%) prepared by mix-
ing 9.5 mL trifluoroacetic acid (TFA), 0.25 mL H2O,
0.25 mL triisopropylsilane (TIS). This cleavage cocktail
is used for the cleavage of linear synthesized tetrameric
peptide and maleimido activated cores.
(b) TFA: EDT:H2O:TIS (94%:2.5%:2.5%:1%) prepared by
mixing 9.4 mL TFA, 0.25 mL ethanedithiol (EDT),
0.25 mL H2O, 0.1 mL TIS. This cocktail is employed for
peptides containing a cysteine residue.
8. Diethyl ether.
9. Dichloromethane (DCM).
10. Dimethylformamide (see Note 8).
11. 3-Maleimidopropionic acid (Sigma-Aldrich Inc, St. Louis, MO).
1. PBS containing 0.01 M EDTA.
2. Argon for flushing solutions.
3. TFA/Anisole mixture (99:1).
4. Silver acetate (Sigma-Aldrich or other vendor).
5. Diethyl ether.
6. Dithiothreitol (0.2 M) prepared in 1 M acetic acid.
7. Guanidine hydrochloride (8 M).
1. HPLC solvent delivery system with binary gradient capabil-
ity and a UV detector.
2. Reversed-phase octadecylsilica (C18) column. In our
laboratory, we use the following columns: Preparative
column: Vydac RP-C18 column (250 mm length×22 mm
diameter, 10 μm particle size). Analytical column: Varian
RP-C18 column (250 mm length×4.6 mm diameter, 5 μm
2.7. Removal of Group
Residues (Outlined in
Purification and Char-
in Subheading 3.13)
296 McGuire, Li, and Brown
3. Solvent filtration apparatus equipped with a 0.45 μm Teflon
filter (Such as Ultra-ware filter apparatus 300/1,000 mL
from Kontes glass company and 0.45 μm Teflon filters from
4. Syringe driven filter units, 0.22 μm porosity, 13 mm (Mil-
5. Eluent A: H2O/0.1% TFA and eluent B: acetonitrile/0.1%TFA.
6. Access to mass spectrometer and/or peptide sequencing facility.
1. Selected phage clone and corresponding synthetic peptide.
2. Reagents indicated in Subheading 2.5.
1. Synthetic peptide prepared with incorporation of biotin.
2. Streptavidin-conjugated Qdots (emission wavelengths cho-
sen to match microscope emission filter set).
3. Microscope slides and coverslips.
4. Chamber slides (8-well) (for example, VWR, Inc., catalog #
62407–296) can be used to culture adherent cells prior to
5. Prolong® Gold antifade reagent with DAPI (Invitrogen).
6. Fluorescence microscope.
7. Streptavidin-coated magnetic beads (Dynabeads M280-SA,
Invitrogen, 6.7 × 108 beads/mL, 1 mg beads binds 700 pmoles
8. Cell isolation magnet (or strong magnet).
9. Streptavidin-phycoerythrin or streptavidin-FITC.
10. Flow cytometer (Cell Lab Quanta, Beckman Coulter or
other suitable instrument).
12. 0.4% Formalin: 37% formaldehyde, Sigma Chemical, diluted
1:10 in PBS, immediately prior to use.
13. Ethanol (70%).
14. Fingernail polish (any color).
The following information is based on our protocols for selec-
tion of peptide ligands for cell recognition and delivery. Selection
2.9. Inhibition of
Selected Phage Clone
by Cognate Peptide
2.10. Applications of
Biopanning of Phage Displayed Peptide Libraries 297
of a peptide ligand, using our protocol, should be expected to
take 5–6 rounds of biopanning. During each round, cells are
incubated with a mixture of phage displaying different peptides.
Phage that do not bind or bind only to the surface of the cell
are washed away. Phage that bind to the cell and are internalized
by the cell are retained. These cell-internalized phage are ampli-
fied in bacteria, isolated, and used as the input in the next round
of biopanning. In each round of selection, the diversity of the
phage sample is reduced while the proportion of phage displaying
a peptide that mediates cell-specific binding is increased.
Once a phage displayed peptide has been selected using the
biopanning protocol, we characterize the binding selectivity and
cell specificity of that phage clone. Our determination of selectiv-
ity compares the binding and uptake of a cell-selected phage clone
with binding and uptake of a control phage clone that was ran-
domly selected from the library. This provides a rough estimate of
the affinity of the peptide. Additionally, it assures that the cellular
binding is due to the selected peptide and is not the result of non-
specific phage binding. The measurement of cell specificity involves
comparison of the selectivity index of a specific phage over a vari-
ety of different cell types. During the characterization process, we
also prepare chemically synthesized versions of the specific peptide,
monomeric and tetrameric, and test the utility of these constructs
as cell-binding reagents out of the context of phage presentation.
Depending on the down-stream applications of the ligand, we will
incorporate a unique cysteine for chemical modification or a biotin
moiety for use with streptavidin-based reagents.
1. Cells are seeded onto tissue culture wells 24–48 h before
panning. Only a single well is required for each panning
round. Once started, each round of the panning procedure
requires approximately 4–5 h to complete. Additional time
is required for bacterial plating for titer determination and
2. 24–48 h before the phage biopanning will be conducted,
trypsinize cells from a propagation flask and seed cells in
12-well plate. On the day of panning, one well should be
≈90% confluent. The proper level of confluence is generally
obtained by seeding 100,000–150,000 cells in a well.
3. Begin the biopanning procedure by gently removing media
from the well. Wash cells with 1 mL RPMI media (or other
cell-appropriate media) without serum (tip plate to accumu-
late liquid on one side of the well so that media can be aspi-
rated without disturbing attached cells. Pipette wash media
gently to avoid dislodging cells. Remove wash media.
4. Gently add 1 mL/well media without serum and incubate
cells for 2 h at 37°C to clear cell surface receptors (referred
to as “clearing the receptors”).
Panning for Adherent
298 McGuire, Li, and Brown
5. Approximately 15 min before the end of the clearing step,
prepare the phage panning solution as follows:
(a) Chloroquine (10 μL) (100× stock).
(b) 40 μL protease inhibitor without EDTA (25× stock).
(c) 10–100 library equivalents of the phage library. The
phage library used for much of our work has a diversity
of 1×108 members (5, 19). Therefore, we add 1×109 –
1×1010 phage to the input sample for round one. Thus,
each library member should be present in 10–100 cop-
ies in the input mixture. For each successive round of
biopanning we input ∼1.5×109 phage.
(d) Bring mixture to 1 mL final volume by addition of PBS+
with 0.1% BSA.
6. Remove RPMI from the cells. Wash cells once with 1 mL
PBS+ with 0.1% BSA that was prewarmed to 37°C. Remove
the wash solution from the cells.
7. Save 50 μL of the input phage solution for titer determina-
tion. Add the remainder of the phage solution to the cells
and incubate for 1 h at 37°C in a standard tissue culture CO2
8. After the 1 h incubation, aspirate the supernatant. We do not
save this solution containing unbound phage. Wash the cells
four times at room temperature:
(a) Add 1 mL PBS+ with 0.1% BSA (room temperature)
(b) Incubate for 5 min.
(c) Aspirate buffer and repeat.
9. Acid elute/wash 1–2 times at room temperature:
(a) Add 1 mL 0.1 M HCl–glycine, pH 2.2 + 0.9% NaCl.
(b) Incubate for 5 min. Time could be reduced if the cell
line is fragile and lysis is problematic (see Note 9).
(c) If you are interested in phage that bind to the cell sur-
face but are not internalized, you can keep this acid wash
fraction when it is removed from the cells and amplify
the recovered phage as detailed below. Adjust the pH of
the acid wash material by addition of 1.5 M Tris–HCl,
pH 8.8 after it is removed from cells.
(d) Repeat acid wash once.
10. Remove the second acid wash and add 1 mL of 30 mM Tris–
HCl, pH 8.0 to the cells and incubate on ice for 30 min. This
hypotonic media is used to swell the cells and enhance lysis
and recovery of phage.
11. Freeze cells in plate. This is a suitable place to stop the pro-
tocol if needed.
Biopanning of Phage Displayed Peptide Libraries 299
12. Thaw cells and scrape off plate. The freeze-thaw cycle dis-
rupts the cells and releases any internalized phage. This sam-
ple is referred to as the output fraction. Examine the well
under a microscope to ensure that the cells have been dis-
rupted. If freeze-thaw does not disrupt the cells, 0.1% Triton
X-100 or other detergent can be added to the hypotonic
13. Set up amplification of output phage as well as titration of
input and output phage. Titer input, acid wash (if desired),
and output. Amplify output or acid wash (if desired).
1. The panning procedure requires approximately 4 h to com-
plete. The cells can be removed directly from a feeder flask
for the panning procedure. They do not have to be seeded
into a separate flask prior to the day of the panning proce-
2. Transfer cells from feeder flask to a 50-mL centrifuge tube
and pellet cells (Speed and time required for forming a good
pellet will vary with cell type).
3. Resuspend cells in 10 mL media without serum and pellet
4. Resuspend cells in 10 mL media without serum and incubate
cells for 2 h at 37°C incubator to clear the receptors.
5. Count cells during clearing and determine volume needed
for 2 million cells.
6. Approximately 15 min before the end of the clearing step,
prepare the phage solution as detailed in step 5 of Subhead-
7. Pellet 2 million cells and wash one time with 10 mL PBS+
with 0.1% BSA pre-warmed to 37°C.
8. Save ∼50 μL input solution for titer determination. Pellet
cells and resuspend the cell pellet gently in the remainder of
phage solution and incubate for 1 h at 37°C in tissue culture
incubator with 5% CO2.
9. At the end of the incubation, dilute the sample to 10 mL
with PBS+ with 0.1% BSA (room temperature).
10. Wash the cells four times by centrifugation and resuspension.
Resuspend cells in 10 mL PBS+ with 0.1% BSA and incubate
for 5 min at room temperature. Pellet cells.
11. Acid wash cells at room temperature:
(a) Resuspend cell pellet in 1 mL 0.1 M HCl–glycine, pH
2.2, 0.9% NaCl.
(b) Incubate 5 min (see Note 9).
(c) Pellet cells and remove supernatant.
3.2. Phage Panning for
300 McGuire, Li, and Brown
(d) Repeat acid wash once. Remove supernatant solution.
12. Remove the second acid wash and add 1 mL of 30 mM Tris–
HCl, pH 8.0 to the cells and incubate on ice for 30 min.
13. Freeze cells and thaw. The freeze-thaw cycle disrupts the cell and
releases any phage. This is referred to as the output fraction.
14. Set up amplification and titration of phage. Titer input, acid
wash (if desired), and output. Amplify output and/or acid
wash (if desired).
1. Between successive rounds of phage biopanning, the output
phage sample (and/or the acid wash sample, if desired) must
be amplified. This procedure may be performed in parallel
with the phage titering as detailed in Subheading 3.3 or
may be performed independently.
2. On day one, pick a single K91 bacterial colony and inoculate
10–15 mL LB media without antibiotics for each sample that
will be amplified.
3. Culture bacteria at 37°C with shaking until an OD600 nm of
0.2–0.4 is obtained.
4. Spin down bacterial cells at 3,000 × g for 10 min at 4°C.
5. Resuspend the pellet in 1/10 the original volume using LB
media by pipetting up and down.
6. Add your phage sample to be amplified (the entire phage
sample – 50 μL aliquot removed for titration) to resuspended
K91 cells and incubate for 15 min at 37°C.
7. Dispense the complete mixture of phage-infected K91 cells
onto four, 150 mm LB-tet plates. Plate 1/4 of the bacterial
8. Allow liquid to be absorbed into plate.
9. Invert plates and incubate at 37°C overnight.
10. On day two, harvest phage. Add 10 mL LB media to each of the
four LB-tet plates. Incubate for 10 min at room tempe-rature.
11. Scrape bacteria off the plate with a glass spreader. Collect all
the material from the four inoculated plates in a single 50 mL
centrifuge tube. Some of the media will not be recovered
from the plate.
12. Add 10 mL fresh LB media to one of the four plates. Use the
glass spreader to clean the plate further and transfer the wash
material to the second plate. Continue until all four plates
have been washed in this manner. Combine this wash mate-
rial with the original harvest in the 50 mL tube.
13. Centrifuge the harvested material to obtain a firm pellet of
bacterial cells (Example: 3,000 × g for 10 min at 4°C in Beckman
Coulter Allegra® 25R centrifuge.).
3.2. Phage Panning for
Biopanning of Phage Displayed Peptide Libraries 301
14. The infectious phage particles will be in the supernatant from
this centrifugation step. Transfer the supernatant to a fresh
centrifuge tube and measure the volume.
15. Add ¼ of the supernatant volume of 2.5 M NaCl + 20% PEG
8000 to the supernatant. Example: if volume of supernatant
is 40 mL, add 10 mL of the NaCl/PEG solution. Incubate
this mixture on ice for 1 h to precipitate the phage particles.
16. Collect the phage precipitate by centrifugation at 11,000 ×
g for 30 min at 4°C. The phage should produce a firm pellet
under these conditions.
17. Pour off and discard the supernatant making sure no stand-
ing liquid is left in the centrifuge tube. Tilt the centrifuge
tube to drain off any residual PEG solution. Leave the tube
inverted for 1 h at 4°C. Residual PEG solution will make it
more difficult to completely resuspend the phage pellet.
18. After draining for 1 h, put the tube upright and add 1 mL
PBS to the pellet. Incubate on ice for 30 min. During this
incubation, tilt the tube so that the pellet is completely cov-
ered with PBS.
19. Gently resuspend the phage pellet using a 1 mL pipette.
Do not vortex. Vortexing concentrated phage solution may
result in shearing of the phage. Mix the samples so that there
are no visible chunks or cakes in the sample. Transfer the
resuspended phage to a clean 1.5 mL microcentrifuge tube.
20. Pellet insoluble debris by centrifugation at 16,000 × g for
2 min in a bench top microcentrifuge.
21. Transfer supernatant to a fresh microcentrifuge tube and
incubate at 65°C for 15 min in a water bath or heating block
to kill any bacteria remaining in the sample. Do not extend
time of this incubation or the phage will lose infectivity.
22. Pellet insoluble debris by centrifugation at 16,000 × g for 2 min.
23. Transfer supernatant to a clean tube. Discard pellet. Mix and
dispense aliquots of the purified phage to clean microcen-
trifuge tubes. Label tube with the cell type, panning round
number, date and operator’s initials.
24. Store aliquots of the phage preparation at −80°C until use.
25. Before using on cells, set up bacterial titration to determine
the yield of infective phage. The titration should be per-
formed as detailed in Subheading 3.3 for the input phage
sample except that more dilute samples are required to infect
with bacteria. We typically dilute amplified phage prepara-
tions 10−2, 10−4, 10−6, 10−7, and10−8. The samples diluted
10−6, 10−7, and10−8 are used to infect K91 bacterial cells and
aliquots of these infections are plated.
26. For amplification of individual phage clones (see Note 10).
302 McGuire, Li, and Brown
1. We maintain K91 cells on minimal media minus proline sup-
plemented with 0.5 μg/mL thiamine. The bacteria grow
slowly on these plates, generally requiring 2 days of culture
to produce suitable colonies. Each day that a titration will
be performed, start a liquid culture of the bacteria from a
2. Pick a single colony and inoculate 5–10 mL LB media with-
3. Culture bacteria at 37°C with shaking until an OD600 nm of
0.4 is obtained. If culture goes past an OD600 nm of 0.6, the
culture should be diluted approximately tenfold with LB
media and continue culturing until the proper optical den-
sity is obtained.
4. If bacterial cells are ready before samples that will be titered,
place bacterial cells on ice until needed. If placed on ice,
rewarm the cells to 37°C prior to mixing with phage sam-
5. Prepare serial dilution of input phage sample (50 μL aliquot
was saved for titering):
(a) Add 10 μL input phage to 990 μL LB media (=10−2 dilu-
(b) Add 10 μL of 10−2 input phage dilution to 990 μL LB
media (=10−4 dilution).
(c) Add 100 μL of 10−4 input phage dilution to 900 μL LB
media (=10−5 dilution).
(d) Add 100 μL of 10−5 input phage dilution to 900 μL LB
media (=10−6 dilution).
6. For titration of biopanning output samples:
(a) Mix the freeze-thaw cell lysate well by flicking.
(b) Add 50 μL of cell lysate to 450 μL LB media (=10−1 dilu-
(c) Add 100 μL of 10−1 dilution to 900 μL LB media (=10−2
(d) For the early rounds of panning, these first two dilutions
should be adequate. After round three, an additional
tenfold dilution is suggested.
7. Dispense 900 μL K91 bacterial cells to sterile tubes for
phage infection. For input phage samples, dispense aliq-
uots to be infected with the 10−4, 10−5, and 10−6 dilutions,
respectively. For the output phage samples, dispense
aliquots to be infected with the 10−1 and 10−2 dilutions,
respectively. Dispense one K91 aliquot that will serve as a
3.4. Bacterial cell
Culture and Phage
Titration (See Note 11)
Biopanning of Phage Displayed Peptide Libraries 303
8. Add 100 μL of diluted phage sample to 900 μL K 91 cells.
Incubate at 37°C for 15 min (see Note 12). Label LB-tet
plates for each sample. We inoculate 2 LB-tet plates for each
infected K91 sample, one plate with 100 μL and second plate
with 50 μL.
9. Add 100 μL of each infected K91 sample onto a separate,
labeled LB-tet plate and spread evenly.
10. Add 50 μL of each infected K91 sample onto a separate,
labeled LB-tet plate and spread evenly.
11. Additionally, plate 100 μL of uninfected K91 cells as a con-
tamination control. After overnight incubation, these con-
trol plates should not have any colonies.
12. Allow plates to dry before inverting.
13. Incubate at 37°C overnight to allow colonies to grow.
14. After overnight incubation, count and record the number
of colonies present on each plate. There should not be any
colonies on the uninfected K91 cell plates. If colonies are
present on these plates, the source of contamination needs
to be eliminated and the samples need to be titered again.
15. Calculate titer of each sample. For each plate, use the for-
mula: (# of colonies × dilution factor× 10 for dilution into
K91)/mL of K91 mixture plated = colony forming units
(cfu)/mL in the original sample. Calculate the independent
determinations for each sample and average them to obtain
16. The output titer plates from biopanning round 3 and sub-
sequent rounds are saved for DNA sequence analysis and
determination of phage displayed peptide sequences (see
1. This protocol assumes the use of the BioRad iCycler. Adjust-
ments to the protocol may be required using other devices.
We use the generalized Sybr® green detection system that
does not require the generation of independent-labeled
2. Turn on the lamp, camera, and thermal cycler at least 30 min
before a reaction starts to stabilize per manufacturer’s sug-
3. Prepare standards and samples. Our calibration standard for
titration consists of a series of tenfold dilutions of phage. We
routinely generate this calibration standard line of infectious
phage that range from 100 to 1 × 109 phage/mL. We have also
run ssDNA and dsDNA preparations of phage in q-PCR.
4. Prepare the master mix for the number of reactions needed.
(See Note 13)
304 McGuire, Li, and Brown
Component Volume per sample (µL)
2× IQ SYBR® Green Supermix50
10 µM forward primer3
10 µM reverse primer3
5. Dispense 90 μL aliquots of mastermix to clean PCR tubes (1
aliquot/sample or DNA standard). Add 10 μL of standard
DNA or phage sample to 90 μL of the master mix.
6. Mix the 100 μL complete reaction mixture and dispense the
reaction mixture into three wells of a 96-well plate that is
optically suitable for q-PCR at 25 μL/well using an 8-chan-
7. Spin down the plate to exclude bubbles at the bottom of the
8. Enter the PCR Protocol and Plate Setup files from the iCy-
cler or appropriate software. We use a 3-step protocol for the
individual amplification cycles:
Procedure Temperature (°C)Time
Hot start95 3 min
Denature 9530 s
Anneal55 30 s
Extension72 30 s
Denature before melt curve analysis951 min
Annealing before melt curve analysis55 1 min
Melt curve analysis 0.5 up10 s
(e) The software for the iCycler (and other real-time thermo-
cyclers) will automatically determine the parameters of the
standard line and calculate the values of phage titers for each
sample. Attention should be paid to the threshold parameter
established by the software, the slope of the standard line
(should be close to 3.2–3.3), and the melt curve analysis,
which indicates the specificity of the amplified product. The
PCR products can be evaluated by agarose gel electrophore-
sis if there is a question about amplification specificity.
Biopanning of Phage Displayed Peptide Libraries 305 Download full-text
1. Dispense 50 μL water to 16 PCR tubes.
2. Label and pick 16-well-spaced colonies on a titer plate of
interest (see Note 14).
3. Using a plastic pipette tip or toothpick, stab each colony and
mix it into a different tube prepared in step 1.
4. Heat the samples at 95°C for 5 min to lyse bacteria and dena-
5. Cool the samples while preparing PCR master mix contain-
ing 100 μL 10× PCR buffer without MgCl2, 60 μL 25 mM
MgCl2, 20 μL 10 mM dNTP mix, 20 μL 10 μM forward
primer (fd-tet F1), 20 μL 10 μM reverse primer (fd-tet B1),
730 μL water and 10 μL Taq polymerase.
6. Dispense 48 μL PCR mastermix to 16 new PCR tubes.
7. Add 2 μL of lysed bacterial colony sample to each aliquot of
8. Perform PCR. We use the following protocol:
Hot start 95 2 min
Denature 9530 s
Anneal 5530 s
Extension 7230 s
Final extension 725 min
9. Evaluate PCR products on 1% agarose gel. Single products
of approximately 450 bp are expected.
10. Remove dNTPs and oligonucleotide primers from the
PCR product by combining 20 μL of PCR product with
2 μL exonuclease I and 2 μL shrimp alkaline phosphatase
(SAP). Incubate at 37°C for 30 min followed by 15 min
11. After exonuclease I and SAP treatment, the PCR product
can serve directly as template in a dideoxy terminator DNA
sequencing reaction. We use BigDye® Terminator v3.1 in the
following mixture: 5 μL treated product, 1 μL 10 μM primer
(fd-tet F1), 3 μL 5× reaction buffer (supplied with BigDye®
Terminator), 2 μL BigDye® Terminator Mix, and 9 μL water.
BigDye® reactions are prepared in a 96-well plate.
12. Perform sequencing PCR using the following conditions:
3.6. Colony PCR for