Table 3. Secondary antibody protein array analysis protocol.
| Protocol steps | Objective | Reagent | Time |
|---|---|---|---|
| Protein array
preparation |
Rinse array | 70% (v/v) ethanol | 5 min |
| Remove ethanol and rinse | dH 20 for 2 | 2 min | |
| Wipe off all E. coli colonies | laminar tissue | As appropriate | |
| Wash 1 | Wash off any E. coli debris | TBST-T | 10min (x3) |
| TBS | 2min (x2) | ||
| TBS | 10 min | ||
| Array blocking | Block arrays by shaking | 5% (w/v) Milk Marvel TBS-T | 2h |
| Wash 2 | Wash off any Blocking solution | TBS-T | 15min (x3) |
| Incubate first
antibody |
Rabbit anti-chicken IgY | 1 in 1,000 (no recommended
western dil.) in 2% (w/v) BSA TBS-T |
2h |
| Wash 2 | Wash off any unbound antibody | TBS-T | 15 min (x3) |
| Incubate second
antibody |
Goat anti-rabbit IgG-AP | 1 in 1,000 in 2% (w/v) BSA TBS-T | 2h |
| Wash 3 | Wash off any unbound antibody | TBS-T | 10 min (x2) |
| TBS | 10 min (x2) | ||
| Protein array signal
detection |
Signal generation for array
bound goat anti-rabbit IgG-AP |
AttoPhos AP Fluorescent Substrate
diluted 1 in 8 in AP buffer (1mM MgCl 2, 100mM Tris base, pH 9.5) |
10 min |
| Protein array image acquisition | FujiScanner Fla5100 (Settings Laser:
473, Filter: LPB, Resolution 50μm |
18 min |