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Affinity Selection
General considerations
Each affinity-selection step starts with a mixture of phage, and seeks to select from that
mixture phage whose displayed peptide binds the target receptor, antibody or other binding
molecule (we call such molecules generically “selectors”). These phage are specifically
“captured” by immobilizing the selector on a solid surface (e.g., a plastic petri dish); unbound
phage are washed away and captured phage are eluted (still in infective form), yielding a selected
subset of the original phage mixture that is called an “eluate.” If a second round of affinity
selection is planned, the eluate from the first round must be amplified by infecting the phage into
fresh cells; the amplified eluate is then used as input to another round of selection. Altogether,
two or three rounds of selection usually suffice to select for good binders—assuming, of course,
the initial library contains such binders.
Stringency versus yield
The stringency of affinity selection is more or less opposed to yield, and is controllable in
some degree by the choice of conditions, as will be detailed below. Yield is all-important in the
first round of selection (see below), but in later rounds yield can be sacrificed in the interest of
stringency.
There is a limit to stringency, however. The reason is that there is always a background
yield of non-specifically bound phage; if stringency is set too high, the yield of specifically
captured phage will fall far below the background of non-specifically bound phage, and all
power of discrimination in favor of high affinity is lost.
In practice, because the relationship between selection conditions and stringency is
unknown in advance, it is advisable to explore a range of conditions in the later rounds of
selection. This usually means trying different amounts of biotinylated selector (<1 µg) in both
the one- and two-step selection procedures described below.
Yield is all-important in the first round of selection
It is vital to favor high yield in the very first round of selection, even if this means
reduced stringency. The input in that round consists of all clones in the initial library. Since the
library has many clones, each clone is represented by few particles (~100 TU/clone in a typical
experiment); consequently, if the yield for a binding clone is not high in the first round (>1%,
say), that clone has a good chance of being lost, and of course can never be recovered. In
practice, where possible we carry out the first round using 10 µg biotinylated selector in the
“one-step” selection procedure described below; this procedure gives the highest yields.
By the logic in the previous paragraph, it is also important that the entire eluate from the
first round be amplified to create the input for the second round (see step 13 and the note above
it). Once the phage are amplified, each clone is represented by millions of phage particles, and
no clones will be lost by using only a portion of the amplified first eluate as input to the second
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round of selection. Similarly, each clone in the unamplified eluates from the second and
subsequent rounds of selection is represented by tens of thousands of phage particles (if not
more), and no clones will be lost if only a portion of such an eluate is amplified.
Capture via a biotinylated selector: “one-step” and “two-step” selection
If selector protein is available in relatively pure form, it is convenient to biotinylate it at
accessible amino groups, as described in biotinylation.doc. This allows it to be rapidly and
irreversibly captured on streptavidin-coated petri dishes or ELISA dish wells under nondenaturing conditions, and also facilitates ELISA. (Numerous alternative immobilization
methods are available, but won't be discussed here.)
The biotinylated selector can be used in two ways (details in the next two subsections): in
“one-step” selection, phage are captured by biotinylated selector that has been pre-immobilized
on the surface of a streptavidin-coated petri dish; while in “two-step” selection, phage are reacted
with biotinylated selector in solution, then subsequently captured on a streptavidin-coated dish.
Input phage
The input to the first round of affinity selection is the initial phage display library; the
input to the second and subsequent rounds of infection is the amplified eluate from the previous
round.
The number of input phage must be large enough to adequately represent all the clones of
interest—i.e., clones with high affinity for the target selector. Since a high-affinity clone
typically gives a yield of ~1% in the affinity-selection process, that means that every clone of
interest should be represented by at least ~100 TU (equivalent typically to ~2000 virions). This
requirement is most difficult to meet in the first round of selection from the initial phage display
library, in which all clones in the primary library—including the clones of interest—are about
equally represented. For a library with 109 primary clones, for instance, the input should have at
least 1011 TU (~2 × 1012 virions), and at least ten times that number if feasible. In contrast, in the
inputs to the second and subsequent rounds of affinity selection (i.e., the amplified eluates from
the previous rounds), the clones of interest are overrepresented and the total number of TUs can
be reduced if necessary without compromising the goals of the project. There is no reason in
most circumstances for the input phage to be purified in any way; indeed, we sometimes use the
culture supernatant from eluate amplification directly as the input to the next round of affinity
selection.
Internal enrichment control phage
Phage fd-cat is described in VECTORS.DOC. Its 7775-base genome is derived from
fd-tet by replacing the tetracycline resistance determinant with the chloramphenicol acetyl
transferase (cat) gene from plasmid vector pBR328. It has the same replication defect as fd-tet;
its infectivity is roughly 5 times less on average (typically ~1%). These phage are propagated
and titered (TUtiter.doc) the same way as fd-tet-derived phage, using chloramphenicol (34 µg/ml
in both plates and liquid medium) in place of tetracycline.
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Since fd-cat virions display no foreign peptides and can be titered independently of fdtet-derived library phage, they can serve as an internal indicator of non-specific background yield
during affinity selection. Thus fd-cat virions are mixed with the input phage (usually equal
numbers of virions of each type) prior to each round of affinity selection. Then, by comparing
the yield of library phage (tetracycline TU) with the yield of fd-cat control phage
(chloramphenicol TU) in the eluates (step 13–17 below), the progress of enrichment at each
round of affinity selection can be quantified. Because substantial numbers of fd-cat control
virions are added to the input of each round of affinity selection, they should be propagated and
purified on a large (e.g., 2-liter) scale (VirionPurification.DOC, without detergent treatment).
Eluates must be amplified before serving as inputs to subsequent rounds of affinity selection
If the eluate (output) from a round of affinity selection is to serve as the input to a
subsequent round, it must first be amplified. There are two reasons for this. First, and most
obviously, amplification is necessary to counter the low yield generally attained in affinity
selection (typically only ~1% for a phage clone displaying a peptide with high affinity for the
selector). Second, and less obviously, the elution process can cause subtle physical damage to the
virions even if it doesn’t impair their infectivity substantially. Such damage can substantially
increase non-specific background yield and thus slow the progress of enrichment for the desired
selector-binding clones.
Amplification is achieved by infecting the eluate phage en masse into a large excess of
fresh cells, propagating those cells in the presence of tetracycline (to select against uninfected
cells), and partially purifying the secreted virions. If the eluate contains fd-cat internal control
phage (conferring resistance to chloramphenicol; see the previous subsection) as well as library
phage (conferring resistance to tetracycline), the former will be strongly selected against during
propagation in the presence of tetracycline. There may be a few residual fd-cat phage in the
amplified eluate because of the freeloader effect (see VECTORS.DOC), but such contaminants
will be negligible compared to the fd-cat control phage that are purposely added prior to a
subsequent round of affinity selection.
One-step selection
NOTE: When the amount of biotinylated selector added at step 4 below is enough to saturate the
immobilized streptavidin (1–10 µg per 35-mm dish), this procedure gives the maximum
achievable yield, which can reach 20% of the input phage. When each phage particle displays
multiple copies of the random peptide, as in most of our libraries, this high yield is plausibly
attributed to attachment of a single virion to two or more neighboring selector molecules (or, in
the case of antibodies, to both Fab domains of a single IgG molecule); a particle captured
multivalently in this fashion may dissociate from the solid surface exceedingly slowly, even if
the underlying monovalent affinity is only modest. As the density of an immobilized
monovalent selector is decreased, this “avidity effect” is reduced, possibly to the point where
yield from monovalent attachment comes to dominate the output—conditions that should
strongly favor high affinity.
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1. Coat a 35-mm petri dish with 400 µl 10 µg/ml streptavidin in 0.1 M NaHCO3 for at least 1 hr
at room temperature or overnight at 4ºC in a humid box (a sealed plastic box with a damp paper
towel on the bottom).
2. Aspirate out the streptavidin solution and fill the dish to brimming with blocking solution;
allow to sit with lid off for 2 hr at room temperature.
3. Pour the blocking solution back into its container (we continue to re-use it until it becomes
cloudy). Wash five times with TBS/Tween from a squirt-bottle, emptying the dish by aspiration
each time.
4. Add the desired amount of biotinylated selector (0.01–10 µg, the latter being recommended in
the first round) in 400 µl TTDBA; allow the dish to react at least 2 hr at 4°.
5. Wash five times with TBS/Tween as in step 3 to remove unbound selector, and fill the dish
with 400 µl of TTDBA. Add 4 µl 10 mM biotin and rock at room temperature for 10 min in order
to block unoccupied biotin-binding sites on the immobilized streptavidinin.
6. Add the input phage (see “Input phage” in the introduction), premixed with fd-cat internal
enrichment control phage if desired (see “Internal enrichment control phage” in the introduction;
a small amount of the premix should be saved for titering at step 17 below), and rock the dish for
4 hr (usually at 4°, but sometimes at other temperatures). There is no need to remove excess free
biotin: it won’t displace the bound biotinylated selector.
7. Wash the dish ten times with TBS/Tween as in step 3.
8. Elute bound phage from the dish with 400 µl of elution buffer for 10 min on a rocker; transfer
the eluate to a microtube and neutralized by mixing it with 50 µl of 1 M Tris-HCl (pH 9.1). If
the elution buffer is yellow as a result of having 0.1 mg/ml phenol red, neutralization will turn
the color rosé. This is the unamplified eluate. If this is not the last round of selection, amplify
the eluate as detailed in steps 13–22. If it is the last eluate, titer the unamplified eluate and
propagate individual clones as detailed in steps 23–25.
Two-step selection
9. Equilibrate input phage (see “Input phage” in the Introduction), premixed with fd-cat internal
enrichment control phage if desired (see “Internal enrichment control phage” in the introduction;
a small amount of the premix should be saved for titering at step 17 below), overnight at 4º with
the desired amount of biotinylated selector (usually less than 1 µg) in TTDBA (typically ~100
µl).
10. Meanwhile, coat a 35-mm dish with streptavidin as in steps 1–3 above and fill it with 400 µl
TTDBA.
11. Add the reaction mixture (step 9) to the streptavidin-coated dish from the previous step.
Rock for 10 min at room temperature to permit capture by immobilized streptavidin.
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12. Wash and elute the dish as in steps 7–8 above.
NOTE: During the equilibrium step (step 9), selectors (assuming they are monovalent) bind
phage reversibly according to solution-phase equilibrium kinetics. If there is little dissociation
and reassociation during the subsequent 10-min capture step (step 11), the situation at the
beginning of the capture step will largely determine the relative yields of different clones. If, at
the other extreme, selectors dissociate and reassociate very rapidly during the capture step, twostep selection is really equivalent to an abbreviated one-step selection. If desired, reassociation
can be suppressed during the capture step by adding a competitive ligand for the selector at high
concentration. In practice, two-step selection gives considerably lower yields than one-step
selection, even when reassociation is not suppressed.
Quantifying yield and amplifying eluates
NOTE: Eluates that are to serve as input for further rounds of affinity selection are amplified by
propagating the phage in fresh host cells. The eluate from the first round is first concentrated
(step 13) to allow the entire eluate to be amplified (see “Yield is all-important in the first round
of selection” in the Introduction). Eluates from subsequent rounds, in which every clone is
represented by many thousands or millions of phage particles, are used without concentration.
13. (For first-round eluates only) Concentrate the entire first-round eluate and wash it once with
TBS on a Centricon 30-KDa ultrafilter (Amicon) by centrifuging at 5 Krpm in the Sorvall SS34
rotor (with thick-walled rubber adaptor) to gave a final volume of 100 µl.
14. In a 1.5-ml Ep tube mix 100 µl of eluate (the entire eluate if from the first round) and 100 µl
of starved K91BluKan (= K91BK) cells (section A of TUtiter.doc) or K91BK terrific broth
culture (section B of TUtiter.doc); incubate 10–30 min at room temperature.
15. Pipette the infected cells into a 250-ml culture flask containing 40 ml NZY with 0.2 µg/ml
tetracycline1; shake 30–60 min at 37°.
16. Spread 200-µl portions of appropriate serial dilutions of the culture (diluent = NZY) on NZY
plates containing 40 µg/ml tetracycline and 100 µg/ml kanamycin to quantify the output of the
affinity selection. If the input was premixed with fd-cat internal control phage (see step 6 or 9),
also spread 200-µl portions of appropriate serial dilutions on NZY plates containing 34 µg/ml
chloramphenicol and 100 µg/ml kanamycin to quantify the background output.
NOTE: Assuming the input to the affinity selection was ~1011 TU, as recommended, dilutions in
the range of 10-1 to 10-4 will cover yields in the range of 3 × 10-5% (typical non-specific
background) to 1% (the highest yields we ordinarily observe). For the first round, the 10-1 and
10-2 dilutions will almost always suffice. The yield of the added fd-cat internal enrichment
control phage (if included) should remain at background levels at all rounds of affinity selection.
1
This concentration of tetracycline is sub-inhibitory but sufficient to derepress the inducible tetA tetracycline
resistance gene. It does not interfere with titering of fd-cat chloramphenicol TU.
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17. At the same time, titer a suitable serial dilution of the input phage (or of the input
phage/fd-cat premix if relevant) (diluent = TBS/gelatin; dilution should aim at ~105 TU/ml,
equivalent to ~2 × 106 physical particles/ml) by the ordinary analytical titering method (section C
of TUtiter.doc) on the same starved cells or terrific broth culture as were used in step 14. If fdtet internal enrichment control phage were included at step 6 or 9, titer for chloramphenicol TU
as well as tetracycline TU. The yield of affinity selection can be calculated by dividing the total
number of output TU by the total number of input TU.
NOTE: A suitable dilution of an initial library with a physical particle concentration of, say, 2 ×
1014 virions/ml (~1013 TU/ml) is 10-8; suitable dilutions of an amplified eluate with a physical
particle concentration of ~5 × 1013 virions/ml are 10-7 and 10-8.
18. Meanwhile, add 40 µl of 20-mg/ml tetracycline to the 40-ml culture step 15 to bring the total
antibiotic concentration to 20 µg/ml; continue shaking overnight at 37º. Cells harboring fd-cat
internal enrichment control phage, if present, will be killed by the tetracycline, and library phage
should therefore greatly predominate in the culture after overnight growth in that antibiotic (see
“Internal enrichment control phage” in the introduction).
19. Pour the culture into a 50-ml tube and clear it of cells by two 10-min centrifugations at 5 and
8 Krpm in a FiberLite rotor2 at 4º. Pour the doubly-cleared supernatant into a fresh 50-ml tube
and note volume (usually ~35 ml).
NOTE: As noted above (see Input phage in the Introduction), there’s usually no need to purify
the amplified phage in any way in preparation for subsequent rounds of affinity selection.
Nevertheless, we generally purify the virions by two successive PEG precipitations (next three
steps) in the hope that the phage are less likely to lose affinity to contaminating proteases upon
prolonged storage.
20. To the doubly-cleared culture supernatant previous step add 0.15 vol (~5.25 ml) PEG/NaCl
and mix by many inversions; allow phage to precipitate overnight in the refrigerator.
21. Collect the precipitated phage by centrifuging at 12 Krpm 15 min at 4º in the FiberLite rotor;
RRR; dissolve pellet in 1 ml TBS and transfer the solution to a 1.5-ml Ep tube; microfuge 1 min
at top speed to clear insoluble material; carefully transfer the supernatant to a second 1.5-ml Ep
tube.
22. Add 150 µl PEG/NaCl; vortex to mix; refrigerate at least 1 hr; microfuge 5 min at top speed;
RRR; dissolve pellet in 400 µl TBS; microfuge 1 min at top speed to clear undissolved material;
transfer supernatant to a 500-µl Ep tube; store in refrigerator. This is the amplified eluate; the
physical particle concentration should be ~5 × 1013 virions/ml3, regardless of the titer in the
2
These excellent rotors allow you to spin standard disposable 50-ml conical screw-cap centrifuge tubes at 15,000
rpm (not relevant here, though). The pellet collects at the angle rather than the tip, which makes it particularly easy
to free of residual supernatant. If you don’t have this rotor, you can use OakRidge tubes and the Sorvall SS34 rotor
(or equivalent Beckman rotor) instead.
3
It’s a good idea, if convenient, to confirm this estimate empirically by scanning a 1/20 dilution from 240 to 320 nm
and calculating the physical particle concentration as described in AbsorptionSpectrum.doc.
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unamplified eluate; the titer is ~0.5–5 × 1012 TU/ml. Usually we use 100 µl of this amplified
eluate for the next round of affinity selection.
NOTE: If the amplified eluate is to be stored for a long time, it’s a good idea to add NaN3 to a
final concentration of 0.02–0.05% to inhibit microbial growth.
Quantifying yield and propagating clones from the final eluate
NOTE: There is usually no need to amplify the final eluate. Instead, yield is determined by
analytical titering of suitable dilutions of both input and output (the unamplified eluate, step ).
The colonies from the output titering also serve as individual output clones that are propagated
and characterized individually by sequencing and binding assays such as ELISA. If fd-cat
internal enrichment control phage were pre-mixed with input phage at step 6 or 9, titer that
mixture and the unamplified final eluate for both tetracycline and chloramphenicol TU at steps
23–24; propagate only the tetracycline-resistant clones at step 25.
23. Using TBS/gelatin as diluent, make suitable serial dilutions of both the input (see note under
step 17) and the unamplified final eluate (step 8 or 12).
NOTE: Assuming an input of 1011 TU, suitable dilutions of the output (unamplified eluate; see
step 8; volume 450 µl) are 10-1 to 10-5. This will cover yields up to ~5%.
24. Titer these dilutions on starved cells (section B of TUtiter.doc) by the analytical titering
procedure (section C of TUtiter.doc). If fd-tet internal enrichment control phage were included
at step 6 or 9, titer both the input/fd-cat premix and the unamplified eluate for chloramphenicol
TU as well as tetracycline TU. The yield of affinity selection can be calculated by dividing the
total number of output TU by the total number of input TU.
25. Propagate the desired number of well separated output tetracycline-resistant colonies from
the output titering for individual characterization. In most cases, these clones are propagated and
processed on the 7-ml scale and crude double-stranded RF prepared as in RFminiprep2.doc.
Meanwhile, the culture supernatants from the 7-ml propagations can be processed for partially
purified virions as described in SmallScaleVirions.doc; these virions can be used, for example,
for binding assays as described in micropan.doc and ELISA.doc.