4. Use standard panning to isolate a more diverse repertoire of
binders.
5. Use competitive panning to isolate higher affinity or more
selective binders.
6. If a deselection target is used to pre-pan the phage, the negative
selection in panning round 3 should be against the deselection
target.
7. The binding capacity of Pierce NeutrAvidin-coated, high binding capacity, eight-well strips (ThermoFisher Scientific, catalog
no. 15508) is ~60 pmol D-biotin per well; therefore, the biotinylated target is added in excess and can be reduced, if
required.
8. If the method was used to isolate cross-reactive binders by
alternating the target protein between panning rounds, the
phage clones should be tested for binding against each of the
different target proteins used.
9. If a deselection target was used during the selection process,
the phage clones should be tested for binding against the
deselection target as a negative control.
10. The binding capacity of Pierce Streptavidin-coated, clear,
96-well (ThermoFisher Scientific, catalog no. 15126) is
~10 pmol D-biotin per well. A concentration of 10 μg/ml of
biotinylated target is usually sufficient to test in a phage ELISA,
but can be optimized, if required.
Acknowledgments
We thank the Biomedical Health Research Centre, University of
Leeds for funding the BioScreening Technology Group, where
these methods were developed.
References
1. Bradbury A, Pluckthun A (2015) Reproducibility: standardize antibodies used in research.
Nature 518(7537):27–29
2. Baker M (2015) Reproducibility crisis: blame it
on
the
antibodies.
Nature
521
(7552):274–276. https://doi.org/10.1038/
521274a
3. McLeod J, Ferrigno PK (2016) Antibody alternatives. Scientist 30:38–45
4. Binz HK et al (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus
ankyrin repeat proteins. J Mol Biol 332
(2):489–503
5. Koide A et al (1998) The fibronectin type III
domain as a scaffold for novel binding proteins.
J Mol Biol 284(4):1141–1151
6. Woodman R et al (2005) Design and validation
of a neutral protein scaffold for the presentation of peptide aptamers. J Mol Biol 352
(5):1118–1133
7. Stadler LKJ et al (2011) Structure-function
studies of an engineered scaffold protein
derived from Stefin A. II: development and
applications of the SQT variant. Protein Eng
Des Sel 24:751–763
8. Tiede C et al (2014) Adhiron: a stable and
versatile peptide display scaffold for molecular
120
Anna A. S. Tang et al.
binders.
5. Use competitive panning to isolate higher affinity or more
selective binders.
6. If a deselection target is used to pre-pan the phage, the negative
selection in panning round 3 should be against the deselection
target.
7. The binding capacity of Pierce NeutrAvidin-coated, high binding capacity, eight-well strips (ThermoFisher Scientific, catalog
no. 15508) is ~60 pmol D-biotin per well; therefore, the biotinylated target is added in excess and can be reduced, if
required.
8. If the method was used to isolate cross-reactive binders by
alternating the target protein between panning rounds, the
phage clones should be tested for binding against each of the
different target proteins used.
9. If a deselection target was used during the selection process,
the phage clones should be tested for binding against the
deselection target as a negative control.
10. The binding capacity of Pierce Streptavidin-coated, clear,
96-well (ThermoFisher Scientific, catalog no. 15126) is
~10 pmol D-biotin per well. A concentration of 10 μg/ml of
biotinylated target is usually sufficient to test in a phage ELISA,
but can be optimized, if required.
Acknowledgments
We thank the Biomedical Health Research Centre, University of
Leeds for funding the BioScreening Technology Group, where
these methods were developed.
References
1. Bradbury A, Pluckthun A (2015) Reproducibility: standardize antibodies used in research.
Nature 518(7537):27–29
2. Baker M (2015) Reproducibility crisis: blame it
on
the
antibodies.
Nature
521
(7552):274–276. https://doi.org/10.1038/
521274a
3. McLeod J, Ferrigno PK (2016) Antibody alternatives. Scientist 30:38–45
4. Binz HK et al (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus
ankyrin repeat proteins. J Mol Biol 332
(2):489–503
5. Koide A et al (1998) The fibronectin type III
domain as a scaffold for novel binding proteins.
J Mol Biol 284(4):1141–1151
6. Woodman R et al (2005) Design and validation
of a neutral protein scaffold for the presentation of peptide aptamers. J Mol Biol 352
(5):1118–1133
7. Stadler LKJ et al (2011) Structure-function
studies of an engineered scaffold protein
derived from Stefin A. II: development and
applications of the SQT variant. Protein Eng
Des Sel 24:751–763
8. Tiede C et al (2014) Adhiron: a stable and
versatile peptide display scaffold for molecular
120
Anna A. S. Tang et al.