further rounds of panning should be performed (e.g., fourth
round of panning in streptavidin-coated wells). The targetspecific phage clones can be further screened by phage ELISA
(see Subheading 3.2).
3.2 Screening
Selected Phage Clones
by Phage ELISA
3.2.1 Expressing
Selected Phage Clones
in a 96-Well Culture Plate
1. Aliquot 200 μl per well of 2YT medium with 100 μg/ml
carbenicillin into a 96-well V-bottom deep well plate using a
multichannel pipette and sterile reservoir.
2. Pick individual colonies from the final panning round of phage
display to inoculate into the wells—pick 48 colonies or whatever the amount you want to test.
3. Incubate overnight at 37
C, 1050 rpm in an incubating
microplate shaker (e.g., Heidolph Incubator 1000 and
Titramax 1000).
4. Aliquot 200 μl per well of 2YT medium with 100 μg/ml
carbenicillin into a new 96-well V-bottom deep well plate.
5. Transfer 25 μl per well of the overnight cultures into this new
plate and incubate for 1 h at 37
C, 1050 rpm in the incubating
microplate shaker. It is important to keep the remaining culture
plate overnight (store at 4
C for up to a week or use it to
prepare fresh cultures for glycerol stocks).
6. Dilute M13K07 helper phage (titer ~10
14 /ml) 1/1000 in 2YT
medium with 100 μg/ml carbenicillin and add 10 μl per well to
the freshly grown cultures using a multichannel pipette. Incubate for 30 min at 37
C, 450 rpm in the incubating microplate
shaker.
7. Dilute the kanamycin stock (25 mg/ml) 1/20 in 2YT medium
with 100 μg/ml carbenicillin and add 10 μl per well to the
phage-infected cultures using a multichannel pipette. Incubate
overnight at 25
C, 750 rpm in the incubating microplate
shaker.
8. Centrifuge the phage-infected culture plate at 3500 Â g for
10 min.
9. The supernatant contains the phage and can be removed
directly into the ELISA plate to test for binding to target.
3.2.2 Phage ELISA
A phage ELISA is performed to test binding of selected phage
clones to target protein. The phagemid DNA from specific phage
binders can then be extracted and identified by DNA sequencing.
1. Block the streptavidin-coated 96-well plate with 300 μl per well
of 2Â Blocking Buffer. Cover the wells with an adhesive plate
seal and incubate overnight at 37
C.
2. Wash the blocked wells 3Â with 300 μl of PBST, preferably
using a 96-well plate washer.
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Anna A. S. Tang et al.
round of panning in streptavidin-coated wells). The targetspecific phage clones can be further screened by phage ELISA
(see Subheading 3.2).
3.2 Screening
Selected Phage Clones
by Phage ELISA
3.2.1 Expressing
Selected Phage Clones
in a 96-Well Culture Plate
1. Aliquot 200 μl per well of 2YT medium with 100 μg/ml
carbenicillin into a 96-well V-bottom deep well plate using a
multichannel pipette and sterile reservoir.
2. Pick individual colonies from the final panning round of phage
display to inoculate into the wells—pick 48 colonies or whatever the amount you want to test.
3. Incubate overnight at 37
C, 1050 rpm in an incubating
microplate shaker (e.g., Heidolph Incubator 1000 and
Titramax 1000).
4. Aliquot 200 μl per well of 2YT medium with 100 μg/ml
carbenicillin into a new 96-well V-bottom deep well plate.
5. Transfer 25 μl per well of the overnight cultures into this new
plate and incubate for 1 h at 37
C, 1050 rpm in the incubating
microplate shaker. It is important to keep the remaining culture
plate overnight (store at 4
C for up to a week or use it to
prepare fresh cultures for glycerol stocks).
6. Dilute M13K07 helper phage (titer ~10
14 /ml) 1/1000 in 2YT
medium with 100 μg/ml carbenicillin and add 10 μl per well to
the freshly grown cultures using a multichannel pipette. Incubate for 30 min at 37
C, 450 rpm in the incubating microplate
shaker.
7. Dilute the kanamycin stock (25 mg/ml) 1/20 in 2YT medium
with 100 μg/ml carbenicillin and add 10 μl per well to the
phage-infected cultures using a multichannel pipette. Incubate
overnight at 25
C, 750 rpm in the incubating microplate
shaker.
8. Centrifuge the phage-infected culture plate at 3500 Â g for
10 min.
9. The supernatant contains the phage and can be removed
directly into the ELISA plate to test for binding to target.
3.2.2 Phage ELISA
A phage ELISA is performed to test binding of selected phage
clones to target protein. The phagemid DNA from specific phage
binders can then be extracted and identified by DNA sequencing.
1. Block the streptavidin-coated 96-well plate with 300 μl per well
of 2Â Blocking Buffer. Cover the wells with an adhesive plate
seal and incubate overnight at 37
C.
2. Wash the blocked wells 3Â with 300 μl of PBST, preferably
using a 96-well plate washer.
118
Anna A. S. Tang et al.