Table 1.
Description of each round of panning performed with Ph.D.-C7C and Ph.D.-12 Phage Display Peptide Library.
| Round | Solid surface for immobilization of target | Target | Blocking buffer | Amount of phages used to pan (in 100 µL) | Washing buffer | Phage elution |
|---|---|---|---|---|---|---|
| Ph.D.-C7C Phage Display Peptide Library | ||||||
| 1st | Pierce Amine-binding, Maleic Anhydride Activated Plates | GTTYGVCSK-biotin (1 µg/well) | 0.5% PBST (pH 7.2) + 1 M glycine | 2 × 1011 PFU in 0.5% PBST (pH 7.2) | 0.5% PBST (pH 7.2) | competitive elution with GTTYGVCSK-biotin (9 µg) in PBS (pH 7.2) |
| 2nd | Pierce Amine-binding, Maleic Anhydride Activated Plates | GTTYGVCSK-biotin (1 µg/well) | 1% PBST (pH 7.2) + 1 M glycine | 2 × 1011 PFU in 1% PBST (pH 7.2) | 1% PBST (pH 7.2) | competitive elution with GTTYGVCSK-biotin (2 µg) in PBS (pH 7.2) |
| 3rd | CovaLink plates | GTTYGVCSK-biotin (1 µg/well) | CovaBuffer (pH 7.2) + 1 M glycine | 2 × 1011 PFU in CovaBuffer (pH 7.2) + 1 M glycine | CovaBuffer (pH 7.2) + 1% Tween® 20 |
(i) non-related peptide in CovaBuffer (pH 7.2) + 1 M glycine; followed by (ii) competitive elution with GTTYGVCSK-biotin (2 µg) in PBS (pH 7.2) |
| 4th | Pierce Nickel Coated Plates | rDIII (0.3 µg/well) | 0.1% TBST (pH 7.2) + 1% BSA | 2 × 1011 PFU in 0.1% TBST (pH 7.2) + 1% BSA | 0.1% TBST (pH 7.2) + 20 mM imidazole | 0.1% TBST (pH 7.2) + 250 mM imidazole |
| Ph.D.-12 Phage Display Peptide Library | ||||||
| 1st | Pierce Amine-binding, Maleic Anhydride Activated Plates | GTTYGVCSK-biotin (1 µg/well) | 0.5% PBST (pH 7.2) + 1 M glycine | 2 × 1011 PFU in 0.5% PBST (pH 7.2) | 0.5% PBST-20 (pH 7.2) | competitive elution with GTTYGVCSK-biotin (9 µg) in PBS (pH 7.2) |
| 2nd | Pierce Amine-binding, Maleic Anhydride Activated Plates | GTTYGVCSK-biotin (1 µg/well) | 1% PBST (pH 7.2) + 1 M glycine | 2 × 1011 PFU in 1% PBST (pH 7.2) | 1% PBST (pH 7.2) | competitive elution with GTTYGVCSK-biotin (2 µg) in PBS (pH 7.2) |
| 3rd | CovaLink plates | GTTYGVCSK-biotin (1 µg/well) | CovaBuffer (pH 7.2) + 1 M glycine | 2 × 1011 PFU in CovaBuffer (pH 7.2) + 1 M glycine | CovaBuffer (pH 7.2) + 1% Tween® 20 |
(i) non-related peptide in CovaBuffer (pH 7.2) + 1 M glycine; followed by (ii) competitive elution with GTTYGVCSK-biotin (2 µg) in PBS (pH 7.2) |
| 4th | Pierce Nickel Coated Plates | rDIII (0.3 µg/well) | 0.1% TBST (pH 7.2) + 1% BSA | 2 × 1011 PFU in 0.1% TBST (pH 7.2) + 1% BSA | 0.1% TBST (pH 7.2) + 20 mM imidazole | 0.1% TBST (pH 7.2) + 250 mM imidazole |
| 5th | Pierce Nickel Coated Plates | rDIII (0.3 µg/well) | 0.1% TBST (pH 7.2) + 1% BSA | 2 × 1011 PFU in 0.1% TBST (pH 7.2) + 1% BSA | 0.1% TBST (pH 7.2) + 20 mM imidazole | competitive elution with GTTYGVCSK-biotin (2 µg) in PBS (pH 7.2) |
CovaBuffer (2 M NaCl, 40 mM MgSO4.7H2O, 0.05% Tween® 20 in PBS).