3. Immobilization of biotinylated target (see Note 8) and deselection target (if using) (see Note 9). Dilute the biotinylated target
and deselection target (if using) to 10 μg/ml (or other appropriate concentration, see Note 10) with 2Â Blocking Buffer.
Add 50 μl per well of diluted target to the first 6 columns of the
plate and 50 μl per well of deselection target (if using) or
2Â Blocking Buffer to the last six columns of the plate. Incubate for 1 h at room temperature on a vibrating platform
shaker.
4. Wash the wells 3Â with 300 μl of PBST and add 10 μl per well
of 10Â Blocking Buffer into all wells using a multichannel
pipette.
5. Addition of phage clones. Add 40 μl per well of phagecontaining supernatant so that each one is tested against the
target and a negative control well (e.g., phage clone A1 is
added to wells A1 and A7). Incubate for 1 h at room temperature on the vibrating platform shaker.
6. Wash the wells 3Â with 300 μl of PBST, preferably using a
96-well plate washer.
7. Detection of phage with anti-Fd-bacteriophage antibody.
Dilute anti-Fd-bacteriophage-HRP 1/1000 in 2Â Blocking
Buffer and add 50 μl per well. Incubate for 1 h at room
temperature on the vibrating platform shaker.
8. Wash the wells 10Â with 300 μl of PBST, preferably using a
96-well plate washer.
9. TMB detection of HRP. Add 50 μl per well of TMB and allow
to develop. (Note the amount of time the plate is allowed to
develop—usually 2–3 min) Measure absorbance at 620 nm.
10. Optional: To stop the reaction, add 50 μl per well of 0.2 M
H 2 SO 4 . Measure absorbance at 450 nm.
4 Notes
1. A biotinylated deselection target can be immobilized for
pre-panning of phage.
2. The binding capacity of Pierce streptavidin-coated, high binding capacity, eight-well strips (ThermoFisher Scientific, catalog
no. 15501) is ~125 pmol D-biotin per well; therefore, the
biotinylated target is added in excess and can be reduced, if
required.
3. A non-biotinylated deselection target can be added to the
selection, if desired, for deselection of binders to highly homologous proteins or binders to fusion tags.
Isolation of Artificial Binding Proteins
119
and deselection target (if using) to 10 μg/ml (or other appropriate concentration, see Note 10) with 2Â Blocking Buffer.
Add 50 μl per well of diluted target to the first 6 columns of the
plate and 50 μl per well of deselection target (if using) or
2Â Blocking Buffer to the last six columns of the plate. Incubate for 1 h at room temperature on a vibrating platform
shaker.
4. Wash the wells 3Â with 300 μl of PBST and add 10 μl per well
of 10Â Blocking Buffer into all wells using a multichannel
pipette.
5. Addition of phage clones. Add 40 μl per well of phagecontaining supernatant so that each one is tested against the
target and a negative control well (e.g., phage clone A1 is
added to wells A1 and A7). Incubate for 1 h at room temperature on the vibrating platform shaker.
6. Wash the wells 3Â with 300 μl of PBST, preferably using a
96-well plate washer.
7. Detection of phage with anti-Fd-bacteriophage antibody.
Dilute anti-Fd-bacteriophage-HRP 1/1000 in 2Â Blocking
Buffer and add 50 μl per well. Incubate for 1 h at room
temperature on the vibrating platform shaker.
8. Wash the wells 10Â with 300 μl of PBST, preferably using a
96-well plate washer.
9. TMB detection of HRP. Add 50 μl per well of TMB and allow
to develop. (Note the amount of time the plate is allowed to
develop—usually 2–3 min) Measure absorbance at 620 nm.
10. Optional: To stop the reaction, add 50 μl per well of 0.2 M
H 2 SO 4 . Measure absorbance at 450 nm.
4 Notes
1. A biotinylated deselection target can be immobilized for
pre-panning of phage.
2. The binding capacity of Pierce streptavidin-coated, high binding capacity, eight-well strips (ThermoFisher Scientific, catalog
no. 15501) is ~125 pmol D-biotin per well; therefore, the
biotinylated target is added in excess and can be reduced, if
required.
3. A non-biotinylated deselection target can be added to the
selection, if desired, for deselection of binders to highly homologous proteins or binders to fusion tags.
Isolation of Artificial Binding Proteins
119